The heterodimeric transcription factor CLOCK/BMAL1 plays an indispensable role in generating daily rhythms in mammals by occupying the positive limb of the transcription-translation feedback loop of the molecular clock. Throughout the circadian cycle, the mRNA and protein profiles of BMAL1 display robust rhythmic changes without time delays, whereas CLOCK is constitutively expressed. A recent study suggested that BMAL1 mediates the nuclear translocation of CLOCK in a circadian manner and that this BMAL1-dependent translocation provides an additional clock regulatory mechanism by generating periodic availability of the heterodimeric transactivation complex (20
). In addition, it has been demonstrated that the absolute molar concentration of BMAL1 is much lower than that of CLOCK in vivo (23
). On the basis of these findings, it is widely accepted that BMAL1 abundance is rate limiting for the transcriptional activation of CLOCK/BMAL1 heterodimers.
Ironically, however, BMAL1 protein levels reach a trough when BMAL1 is displaying the greatest transcriptional activity and peak during the transcription inhibition phase in diverse tissues and cells, including the SCN (23
) (Fig. ). Furthermore, the decline in BMAL1 at the transcriptional peaks is not due to a reduction of the protein in the cytosol but in the nucleus (Fig. ). To explain these paradoxical results, we have dissected the molecular events underlying transcriptional activation of CLOCK/BMAL1. Our findings lead us to propose the model of the molecular clock shown in Fig. , which we believe accounts for most previous findings.
FIG. 8. Model of the circadian regulation of the degradation of CLOCK/BMAL1 coupled with its functional activation. (A) BMAL1 shuttling promotes the rapid degradation and functional activation of CLOCK/BMAL1 heterodimers during the transcriptionally active stage. (more ...)
During the transcriptional activation stage, BMAL1 shuttling promotes nuclear translocation of CLOCK and E-box-dependent clock gene transcription, coupled with rapid proteolysis of both BMAL1 and CLOCK via ubiquitin-dependent and -independent pathways, respectively (Fig. ). Thus, transcription-coupled protein degradation seems to be sufficient to lead to the BMAL1 protein minimum as well as nuclear depletion of CLOCK/BMAL1 despite the massive de nova synthesis of BMAL1 required for transcriptional activation at the peak of target gene transcription. Indeed, treatment of wild-type MEFs with MG132 at this time dramatically increased BMAL1 levels, whereas CHX treatment reduced them (Fig. ). In contrast, at the transcriptional minimum, the increased levels of the CRY proteins facilitate the binding of CRY to the CLOCK/BMAL1 complex, thereby stabilizing this complex, as well as suppressing its transcriptional activity (Fig. ). This notion is supported by the fact that neither MG132 nor CHX caused a significant change of BMAL1 abundance in the cells at the phase of minimum transcription (Fig. ). Cry-deficient cells, however, had low protein levels relative to their mRNA levels, and this effect was reversed by the exogenous expression of CRY1 and/or CRY2 (Fig. ). Therefore, the increased CRY levels should permit nuclear accumulation of CLOCK/BMAL1 without massive protein synthesis.
Our model emphasizes that transactivation of CLOCK/BMAL1 is tightly coupled with its degradation. This scenario is consistent with the recently proposed “black widow model” (27
). According to this model, most unstable transcription factors, including Jun, Fos, Myc, p53, and HIF1-α, possess TADs that overlap functionally with their degradation signals and control transcription of downstream genes by mechanisms involving the ubiquitin-proteasome system. Processes that limit the activity, location, and abundance of transcription activators might play an important role in keeping their functions tightly in check, and proteolysis could be a major mechanism regulating transcriptional activity. Our results demonstrate that the active complex of BMAL1 and CLOCK promotes rapid proteolysis of both its component proteins in an activation-dependent manner, like a “one shot, one kill” mechanism, although only BMAL1 degradation is mediated by the ubiquitin-proteasome pathway. It might be, therefore, that the rapid and controlled destruction of CLOCK/BMAL1 allows tight control of periodicity by ensuring that activation of the target gene is linked to ongoing synthesis of its transcriptional regulator.
There are some other posttranslational modifications that have been postulated to be required for regulating activity and/or stability of CLOCK/BMAL1, although their functional relevance is still elusive. A prominent example is phosphorylation. Biochemical analysis has revealed that hyperphosphorylation of both proteins occurs in the nucleus but not in the cytoplasm (20
). This event could play a substantial role in the activation-coupled degradation of them. Indeed, several previous studies have demonstrated that phosphorylation is a prerequisite step for the ubiquitin-dependent proteolysis, including PER2 protein (10
). Another possible mechanism implicated in functional regulation of the CLOCK/BMAL1 heterodimer is SUMO modification. Recently, Sassone-Corsi and coworkers have demonstrated that BMAL1 undergoes rhythmic sumoylation in parallel with Per gene transcription, and this process is required for circadian expression of BMAL1 and for its instability (6
). Thus, investigation of the relationship between ubiquitination and sumoylation of BMAL1 could provide insight into the precise mechanisms that control CLOCK/BMAL1-mediated circadian gene activation.
The molecular basis of the mammalian circadian clock has been largely uncovered, but there are several important questions remain unanswered. One such question is what is the mechanism that causes the fundamental feedback loop to oscillate stably with an approximately 24-h periodicity? The general delay model assumes that the most important parameter driving this periodicity is a “time-delay event” that regulates the production of functional clock proteins from their mRNAs (24
). In mammals, both the negative and the positive elements of the feedback loop, Per1, Per2, and Bmal1, exhibit a robust circadian rhythm with respect to mRNA levels, but the rhythmicity of Bmal1 mRNA is antiphase to the oscillations of the Per genes (9
). The time lags between the transcription and translation of the Per genes are around 6 h in rodent tissues and cells (13
). On the basis of the empirical data, a working model for the mammalian clock hypothesizes that that the Bmal1 mRNA rhythm drives the BMAL1 protein rhythm with a 4- to 6-h delay, because this would increase the availability of CLOCK/BMAL1 heterodimers at the time they are required to drive the transcription of the Per and Cry genes (38
). Unexpectedly, however, there is little delay between the appearance of Bmal1 mRNA and BMAL1 protein (23
). Our present findings provide several indications that transcriptional activation of CLOCK/BMAL1 is tightly associated with its degradation. This rapid activation-coupled degradation may alter the apparent BMAL1 profile by masking the absolute amount of newly made BMAL1, thereby filling the temporal gap between synthesis of its RNA and of its protein. Thus, an alternative mechanism explaining how a 24-h time constant is built into the molecular clockwork would include this hidden time delay between the transcription and translation of Bmal1.
Finally, it is important to establish whether the activation-coupled degradation of BMAL1 is essential for the timing keeping system in the SCN. This issue needs to be explored further because we cannot rule out the possibility that there is some functional redundancy within the SCN and not within peripheral clocks; this would allow the SCN to dominate the peripheral oscillators even in the presence of a genetic defect that disrupts normal clock function (29
). However, the fact that BMAL1 levels in the SCN of rats also reach a trough at the time predicted for the peak of Per gene transcription (41
) supports our contention that the rapid degradation of BMAL1 during the transcriptional active phase may be an intrinsic feature of core clockwork throughout the entire organism and reinforces the idea of a hidden time delay between the transcription and translation of Bmal1.
In conclusion, we have demonstrated that BMAL1 shuttles between the cytoplasm and the nucleus, using its functional NLS and NES, to translocate CLOCK to the nucleus and thereby allow dynamic control of CLOCK/BMAL1 transactivation, which is tightly coupled with its degradation via ubiquitin-dependent or -independent pathways. These findings reveal a new aspect of the molecular clock suggesting that the decrease in BMAL1 abundance during the circadian cycle is due to its robust transcriptional activation rather than inhibition of its synthesis.