Molecular basis of the circadian oscillator

The circadian oscillator is based on molecular events that are performed in approximately one day. Once a loop is completed, the system is back to its starting point, and a new cycle can start again. The identification of the molecular components of circadian clocks in

cyanobacteria, fungi, flies, mammals and other organisms indicates that the basic clockwork circuitry in each system may be composed of only a few central components. Although the clock genes are different in different phyla, their expression shares common features and is based on negative feedback loop mechanisms. Thus, the core clock is composed of clock genes encoding positive regulatory proteins, while others encode negative regulatory proteins.

Positive factors activate the transcription of negative factors. The protein levels of the negative factors increases, and at a certain threshold these factors start to attenuate their own expression by interfering with the positive factors acting on their own promoter. As a consequence, the concentration of the negative components decreases until the autorepression can no more be performed. This brings the system back to its starting point. In parallel, the positive factors activate different target genes, whose expression is thus also dependent on the cyclic regulation exerted by the negative factors. These genes are called clock-controlled genes (CCGs). They are not part of the oscillator itself and constitute the basis of the oscillator’s output. The CCGs then influence physiology and behavior of the organism, either directly or indirectly by controlling additional downstream genes (Figure 2).

Inputs into the circadian system are responsible for resetting the phase of the oscillator. Under natural conditions, light and temperature are the dominant Zeitgebers.

However in the laboratory, it was shown that various stimuli could also reset the clock, mostly by a rapid and transient alteration of the expression of the negative core clock genes.

Such prompt responses allow classifying the negative factors in the family of immediate early genes (IEGs) (Albrecht et al., 1997; Balsalobre et al., 2000a; Balsalobre et al., 1998;

Balsalobre et al., 2000b; Shearman et al., 1997; Shigeyoshi et al., 1997).

The scheme in Figure 2 represents a simplified model of the circadian system, which in reality, can be made of one or more interconnected feedback loops. Furthermore, the situation can be even more complex since clock outputs can feed back on the clock itself. And in other cases, the oscillator might also regulate components of input pathways.

Figure 2. Molecular basis of the circadian oscillatory loop. The phase of the cycle is reset by input stimuli. The interplay between positive and negative core clock regulators generates the 24 hours cyclic expression pattern. The same positive and negative elements also drive the expression of clock-controlled genes that are not part of the oscillator itself. In turn, clock-clock-controlled genes drive diverse biological processes making up the clock’s output.

While sharing a similar molecular basis, each organism obviously possesses different clock genes and diverse regulatory mechanisms to generate circadian rhythmicity. The circadian clock has been studied at the biochemical and genetic level in different model organisms, such as the cyanobacterium Synechococcus elongatus, the filamentous fungus Neurospora crassa, the fruit fly Drosophila melanogaster, the mouse Mus musculus, the microalgae Ostreococcus tauri, and the plant Arabidopsis thaliana. The identified positive elements include KaiA (kai means cycle in Japanese) in S.elongatus, WC-1 and WC-2 (white collar 1 and 2) in N.crassa, dCLK (clock) and CYC (cycle) in Drosophila, mCLK and mBMAL1 (brain and muscle Arnt-like protein 1) in mice, and APRR1 (pseudo-response regulator 1, also known as TOC1) in O.tauri and A.thaliana. In these six systems, the negative elements are KaiC in S.elongatus, FRQ (frequency) in N.crassa, dPER (period) and dTIM (timeless) in Drosophila, mPER1, mPER2, mPER3, and mCRY1, mCRY2 (cryptochrome 1 and 2) in mice, and CCA1 (circadian and clock associated 1) in O.tauri accompanied by LHY (late elongated hypocotyl) in A.thaliana (Corellou et al., 2009; Hardin,

transcriptional feedback loops associated with each of this model organisms are described in more details in sections 2.3 and 2.4.

Although rhythmic transcription seems to be required for rhythm generation by the core oscillator, it is becoming clear that post-transcriptional mechanisms also have important roles in producing the appropriate circadian expression profiles of some core clock components in most organisms (Dibner et al., 2009; Harmer et al., 2001; Kojima et al., 2011;

Mehra et al., 2009; O'Neill et al., 2011; Tomita et al., 2005). Post-transcriptional regulation can take place at the level of the mRNA; for example the transcription of Drosophila period gene (dper) is less rhythmic than the accumulation of its mRNAs. This suggests that a regulation at the level of mRNAs stability (modulated by differential splicing) contributes to the observed circadian accumulation (Majercak et al., 2004; Suri et al., 1999). Translational control can provide another stage of control; in Neurospora, for example, alternative initiation of translation and daytime-specific phosphorylation of the essential clock protein frequency (FRQ) give rise to two forms of FRQ proteins and drive their turnover; these controls of the ratio and abundance of FRQ isoforms turned out to be required for precise clock operation (Garceau et al., 1997). Changes in the subcellular localization of mammalian period proteins (mPERs) were also revealed to be important parameters for progression of the clockwork cycle. This subcellular gating was shown to involve post-translational modifications as well as protein-protein interactions (Vielhaber et al., 2001). Finally, protein stability also represents an important regulatory step in maintaining the robustness of the circadian clock.

Mammalian period 2 protein (mPER2) acetylation and Bmal1 protein (mBMAL1) phosphorylation have been shown to promote their stabilization and degradation, respectively (Asher et al., 2008; Sahar et al., 2010). So these multiple levels of post-transcriptional controls are in some cases essential for clock function, presumably to delay the steps between transcription and protein activity and thereby to prevent that the system collapses to equilibrium. In others cases, they are built into this system to maintain robust amplitude of

cycling, to buffer the clock mechanism against abrupt changes or to provide mechanisms by which the clock can be reset by environmental input.