Summary Circadian rhythms in mammals are generated by a transcriptional negative feedback loop that is driven primarily by oscillations of PER and CRY, which inhibit their own transcriptional activators, CLOCK and BMAL1. Current models posit that CRY is the dominant repressor while PER may play an accessory role. In this study, however, constitutive expression of PER, and not CRY1, severely disrupted the clock in fibroblasts and liver. Furthermore, constitutive expression of PER2 in the brain and SCN of transgenic mice caused a complete loss of behavioral circadian rhythms in a conditional and reversible manner. These results demonstrate that rhythmic levels of PER2, rather than CRY1, are critical for circadian oscillations in cells and in the intact organism. Biochemical evidence supports an elegant mechanism for the disparity: PER2 directly and rhythmically binds to CLOCK:BMAL1, while CRY only interacts indirectly; PER2 bridges CRY and CLOCK:BMAL1 to drive the circadian negative feedback loop.

Circadian rhythms in mammals are generated by a negative transcriptional feedback loop in which PERIOD (PER) is rate-limiting for feedback inhibition. Casein kinases I␦ and I (CKI␦/) can regulate temporal abundance/activity of PER by phosphorylation-mediated degradation and cellular localization. Despite their potentially crucial effects on PER, it has not been demonstrated in a mammalian system that these kinases play essential roles in circadian rhythm generation as does their homolog in Drosophila. To disrupt both CKI␦/ while avoiding the embryonic lethality of CKI␦ disruption in mice, we used CKI␦-deficient Per2 Luc mouse embryonic fibroblasts (MEFs) and overexpressed a dominant-negative mutant CKI (DN-CKI) in the mutant MEFs. CKI␦-deficient MEFs exhibited a robust circadian rhythm, albeit with a longer period, suggesting that the cells possess a way to compensate for CKI␦ loss. When CKI activity was disrupted by the DN-CKI in the mutant MEFs, circadian bioluminescence rhythms were eliminated and rhythms in endogenous PER abundance and phosphorylation were severely compromised, demonstrating that CKI␦/ are indeed essential kinases for the clockwork. This is further supported by abolition of circadian rhythms when physical interaction between PER and CKI␦/ was disrupted by overexpressing the CKI␦/ binding domain of PER2 (CKBD-P2). Interestingly, CKBD-P2 overexpression led to dramatically low levels of endogenous PER, while PER-binding, kinase-inactive DN-CKI did not, suggesting that CKI␦/ may have a non-catalytic role in stabilizing PER. Our results show that an essential role of CKI␦/ is conserved between Drosophila and mammals, but CKI␦/ and DBT may have divergent noncatalytic functions in the clockwork as well.casein kinase I delta ͉ casein kinase I epsilon ͉ dominant-negative mutant ͉ PERIOD

Mounting evidence suggests that PERIOD (PER) proteins play a central role in setting the speed (period) and phase of the circadian clock. Pharmacological and genetic studies have shown that changes in PER phosphorylation kinetics are associated with changes in circadian rhythm period and phase, which can lead to sleep disorders such as Familial Advanced Sleep Phase Syndrome in humans. We and others have shown that casein kinase 1δ and ε (CK1δ/ε) are essential PER kinases, but it is clear that additional, unknown mechanisms are also crucial for regulating the kinetics of PER phosphorylation. Here we report that circadian periodicity is determined primarily through PER phosphorylation kinetics set by the balance between CK1δ/ε and protein phosphatase 1 (PP1). In CK1δ/ε-deficient cells, PER phosphorylation is severely compromised and nonrhythmic, and the PER proteins are constitutively cytoplasmic. However, when PP1 is disrupted, PER phosphorylation is dramatically accelerated; the same effect is not seen when PP2A is disrupted. Our work demonstrates that the speed and rhythmicity of PER phosphorylation are controlled by the balance between CK1δ/ε and PP1, which in turn determines the period of the circadian oscillator. Thus, our findings provide clear insights into the molecular basis of how the period and phase of our daily rhythms are determined.dynamic regulation of phosphorylation | stoichiometry | period determination

Background Circadian clocks coordinate an organism’s activities and regulate metabolic homeostasis in relation to daily environmental changes, most notably light/dark cycles. As in other organisms, the timekeeping mechanism in mammals depends on a self-sustaining transcriptional negative feedback loop with a built-in time delay in feedback inhibition. Although the time delay is essential for generating a slow, self-sustaining negative feedback loop with a period close to 24 hours, the exact mechanisms underlying the time delay are not known. Results We show here that RNA interference mediated by microRNAs (miRNAs) is an essential mechanism in generating the time delay. In Dicer-deficient (and thus miRNA-deficient) cells and mice, circadian rhythms were dramatically shortened (by ~2 hours), although the rhythms remained robust. The period shortening was caused by faster PER1 and PER2 translation in the Dicer-deficient cells. We also identified three specific miRNAs that regulate Per expression, and showed that knockdown of these miRNAs in wild-type cells also shortened the circadian period. Conclusions Consistent with the canonical function of miRNAs as translational modulators of target genes and their widespread roles in cell physiology, circadian rhythms are also modulated by miRNA-mediated RNA interference acting on posttranscriptional regulation of key clock genes. Our present study definitively shows that RNA interference is an important modulator of circadian rhythms by controlling the pace of PER synthesis, and presents a novel layer of regulation for the clock.

The mammalian circadian oscillator is primarily driven by an essential negative feedback loop comprising a positive component, the CLOCK-BMAL1 complex, and a negative component, the PER-CRY complex. Numerous studies suggest that feedback inhibition of CLOCK-BMAL1 is mediated by timedependent physical interaction with its direct target gene products PER and CRY, suggesting that the ratio between the negative and positive complexes must be important for the molecular oscillator and rhythm generation. We explored this idea by altering expression of clock components in fibroblasts derived from Per2Luc and Per mutant mice, a cell system extensively used to study in vivo clock mechanisms. Our data demonstrate that the stoichiometric relationship between clock components is critical for the robustness of circadian rhythms and provide insights into the mechanistic organization of the negative feedback loop. Our findings may explain why certain mutant mice or cells are arrhythmic, whereas others are rhythmic, and suggest that robustness of circadian rhythms can be increased even in wild-type cells by modulating the stoichiometry.Sleep/wake cycles and other mammalian circadian rhythms are synchronized with changes in the local environment, most notably light/dark cycles, through endogenous circadian clocks (1-5). A master clock is located in the suprachiasmatic nuclei in the anterior hypothalamus; this clock adjusts itself based on light/dark information and synchronizes peripheral clocks present in most tissues. The molecular composition and operating mechanism of the clocks are very similar, if not identical, among suprachiasmatic nuclei and peripheral tissues (6, 7).The cell-autonomous molecular clock consists of several interacting transcriptional/post-translational feedback loops (8, 9). However, as found in most organisms, including Neurospora, Drosophila, and mammals (1, 10 -12), one negative feedback loop seems to be the primary driver of clock function; this loop is composed of positive elements and negative elements. In mammalian clock cells, CLOCK (or NPAS2) and BMAL1 are the positive elements, and they form a heterodimer that activates transcription of the negative components PER and CRY, which then constitute an inhibitory complex. The inhibitory complex closes the negative feedback loop by inhibiting the positive complex (CLOCK-BMAL1) through direct physical interaction (3, 4, 13-18). Although CLOCK and BMAL1 are dynamically regulated at the posttranslational level in a circadian fashion (14, 19 -22), their oscillations in abundance do not seem to be required for clock function (15,23,24). However, oscillations of the negative complex are critical for the clock, and PER seems to be ratelimiting for the rhythmic formation of the complex (14, 15). Constitutive overexpression of PER leads to constitutively elevated levels of the negative complex and constitutive down-regulation of CLOCK-BMAL1-controlled genes (15).Although the precise mechanism of the inhibition by the negative complex is not known, the mode of...