Mammalian homologs of Drosophila period have been cloned; like period, they lack the bHLH domain that characterizes the Clock protein (see below) cloned from mammals. The human and mouse genes (hPER and mPer, respectively) encoding PAS-domain-containing polypeptides are described. They are highly homologous to Drosophila Per. (PAS is a dimerization domain present in Per, Amt and Sim). In addition to conserved PAS domains, the fly and mammalian proteins contain an amino-terminal homologous stretch containing conserved nuclear localization sequences followed by the two PAS domains. A short segment between residues 624 and 645 corresponds to the site at which the perS mutation of Drosophila renders a short circadian period. An additional homologous region is found in the carboxy terminus in the Per-C region followed by a serine-glycine repeat and a homologous sequence further downstream from from the repeat. As well as this structural resemblance, mPer shows autonomous circadian oscillation in its expression in the suprachiasmatic nucleus, which is the primary circadian pacemaker in the mammalian brain. Clock, a mammalian clock gene encoding a PAS-containing polypeptide, has now been cloned: it is likely that the Per homologs dimerize with other molecule(s) such as Clock through PAS-PAS interaction in the circadian clock system (Tei, 1997).

A mouse gene, mper1, having all the properties expected of a circadian clock gene, is expressed in a circadian pattern in the suprachiasmatic nucleus (SCN). mper1 maintains this pattern of circadian expression in constant darkness and can be entrained to a new light/dark cycle. A second mammalian gene, mper2, also has these properties and a greater homology to the Drosophila gene period. The overall percent homology between mper2 and Per is 53%, whereas this figure drops to 44% when comparing mper1 and Per. The clock protein, the only mammalian PAS domain protein for which there is functional evidence of involvement in circadian rhythms, is distantly related to the period family members. The mper2 sequences have two helical regions in the N-terminal region, separated by about ten amino acids that have conformations typical for a loop. The mper1 predicted protein has three basic amino acids in the putative basic region, whereas mper2 has only two. Expression of mper1 and mper2 is overlapping but asynchronous by 4 hr. mper1, unlike period and mper2, is expressed rapidly after exposure to light at CT22. It appears that mper1 is the pacemaker component that responds to light and thus mediates photic entrainment (Albrecht, 1997).

A new member of the mammalian period gene family, mPer3, was isolated and its expression pattern characterized in the mouse brain. Like mPer1, mPer2 and Drosophila period, mPer3 has a dimerization PAS domain and a cytoplasmic localization domain. mPer3 transcripts showed a clear circadian rhythm in the suprachiasmatic nucleus (SCN). Expression of mPer3 is not induced by exposure to light at any phase of the clock, distinguishing this gene from mPer1 and mPer2. Cycling expression of mPer3 was also found outside the SCN in the organum vasculosum lamina terminalis (OVLT), a potentially key diencephalic region regulating rhythmic gonadotropin production and pyrogen-induced febrile phenomena. Thus, mPer3 may contribute to pacemaker functions both inside and outside the SCN (Takumi, 1998).

To understand how light might entrain a mammalian circadian clock, the effects of light were examined on mPer1, which exhibits robust rhythmic expression in the SCN. mPer1 is rapidly induced by a single thirty minutes duration exposure to light at levels sufficient to reset the clock; dose-response curves reveal that mPer1 induction shows both reciprocity and a strong correlation with phase shifting of the overt rhythm. Light elicits an aburpt rise in the level of mPer1 transcript; mRNA content rises five- to eight-fold in different experiments and transiently reaches levels typically seen at the peak of the daily cycle in transcrpt abundance before returning to background levels. The next peak in mPer1transcript levels following the pulse is delayed by 2 hours, as compared to controls that have not received light. This shows that the phase of the rhythm can be rapidly reset by light. Thus, in both the phasing of dark expression and the response to light, mPer1 is most similar to the Neurospora clock gene frq. Within the SCN there appears to be localization of the induction phenomenon, consistent with the localization of both light-sensitive and light-insensitive oscillators in this circadian center (Shigeyoshi, 1997).

A mammalian homolog of the Drosophila period gene has been identified and designated as Per2. The PER2 protein shows >40% amino acid identity to the protein of another mammalian per homolog (designated Per1) that has been cloned and characterized. Both PER1 and PER2 proteins share several regions of homology with the Drosophila PER protein, including the protein dimerization PAS domain. Phylogenetic analysis supports the existence of a family of mammalian per genes. In the mouse, Per1 and Per2 RNA levels exhibit circadian rhythms in the SCN and eyes, sites of circadian clocks. Both Per1 and Per2 RNAs in the SCN are increased by light exposure during subjective night but not during subjective day. mPer1 and mPer2 RNA levels in the SCN are differentially regulated by light. The acute photic induction of mPer1 RNA levels is reminiscent of the situation in Neurospora in which light pulses acutely induce frq mRNA levels. The rapid induction of frq by light correlates with the phase-shifting properties of light in fungus. The rapid photic induction of mPer1 RNA levels during subjective night and the phase relationship of mPer1 rhythm to circadian time are both consistent with mPer1 RNA induction, providing a clock-specific event for photic entrainment in the SCN. The delayed induction of mPer2 RNA levels following a light pulse shows a clear dissociation between the regulation of mPer1 and mPer2 RNA levels. This delay suggests that the mPer2 RNA rhythm is secondarily affected by light. Eye rhythms are clearly delayed by 3-6 hours relative to the SCN oscillations; in the eye, mPer1 and mPer2 rhythms are synchronous (Shearman, 1997).

The mouse cDNA of a third mammalian homolog of the Drosophila period gene has been cloned and characterized and designated mPer3. The mPER3 protein shows 37% amino acid identity with mPER1 and mPER2 proteins. The three mammalian PER proteins share several regions of sequence homology, and each contains a protein dimerization PAS domain. Hybridization with the antisense probe reveals highest levels of mPer3 gene expression in the SCN, hippocampus, piriform cortex, and cerebellum. Lower levels of mPer3 RNA are detected in neocortex. mPer3 RNA levels oscillate in the suprachiasmatic nuclei (SCN) and eyes. The phase of the mPer3 RNA rhythm is very similar to the phase of mPer1 and mPer2 rhythms in the SCN. mPer3 also displays a circadian rhythm in RNA abundance in eyes, synchronous with mPer1 and mPer2 eye rhythms. The daily profiles of mPer1, mPer2, and mPer3 gene expression in three peripheral tissues (liver, skeletal muscle, and testis) were examined. Circadian rhythms in RNA abundance are evident in all three tissues, with peak levels centered around CT 9-18 (9 after the light goes on to 6 hours after the light goes off). Rhythms in mPer3 are only clearly found for the 7.0 kb transcript in liver and testis. In skeletal muscle, there were synchronous mPer3 rhythms for both the 7.0 kb and 9.0 kb transcripts. mPer2 RNA levels were not quantified in the testis because of low levels of expression. All peripheral tissues, with the exception of liver, display broad peaks in mPer RNA abundance. Levels of all three mPer RNAs are sharply elevated at CT 15 in liver. In general, the phase of the circadian oscillations in mPer RNA levels in the three peripheral tissues is more similar to that in retina than in the SCN. Acute light regulation of the mPer3 gene in the SCN is strikingly different from that of the mPer1 and mPer2 genes. mPer3 RNA levels are unresponsive to light pulses applied throughout the circadian cycle. This contrasts strongly with the acute photic induction of both mPer1 and mPer2 RNA levels during subjective night (Zylka, 1998a)

Mouse (mTim) and human (hTIM) orthologs of the Drosophila timeless (dtim) gene have been cloned and characterized. The mammalian Tim genes are widely expressed in a variety of tissues; however, unlike Drosophila, mTim mRNA levels do not oscillate in the suprachiasmatic nucleus (SCN) or retina. Importantly, hTIM interacts with the Drosophila PERIOD (dPER) protein as well as the mouse PER1 and PER2 proteins in vitro. In Drosophila (S2) cells, hTIM and dPER interact and translocate into the nucleus. Finally, hTIM and mPER1 specifically inhibit CLOCK-BMAL1-induced transactivation of the mPer1 promoter. Taken together, these results demonstrate that mTim and hTIM are mammalian orthologs of timeless and provide a framework for a basic circadian autoregulatory loop in mammals (Sangoram, 1998).

The mouse cDNA of a mammalian homolog of the Drosophila timeless gene has been isolated. The mTim protein shows five homologous regions with Drosophila TIM. The first conserved region (C1) encompasses the amino-terminal regions of both the mouse and fly proteins. Between C1 and C2 of dTIM, there is a stretch of 223 residues not found in mTIM. mTIM appears to lack the 5' half of the first PER interactive domain defined in dTIM. The second PER interactive domain (IAD-2) of dTIM is present in C2-C4 of mTIM. In mTIM, however, this domain is interrupted by two long stretches of amino acids not present in dTIM. Between C4 and C5 of dTIM, there is a stretch of 175 amino acids not found in mTIM. C5 represents a small area in the carboxyl end of mTIM that is highly conserved among dTIM and mTIM and also in silkmoth TIM. Within the nonconserved region between C2 and C3 of mTIM, there is a stretch of 10 basic amino acids and a stretch of 11 acidic amino acids. An acidic region resides in the nonconserved region of dTIM between C1 and C2. No motifs of structural significance are detected in the mTIM protein. mTim is weakly expressed in the suprachiasmatic nuclei (SCN) but exhibits robust expression in the hypophyseal pars tuberalis (PT). mTim RNA levels do not oscillate in the SCN nor are they acutely altered by light exposure during subjective night. mTim RNA is expressed at low levels in several peripheral tissues, including eyes, and is heavily expressed in spleen and testis. Yeast two-hybrid assays reveal an array of interactions between the various mPER proteins but no mPER-mTIM interactions. The data suggest that PER-PER interactions have replaced the function of PER-TIM dimers in the molecular workings of the mammalian circadian clock. Since mTim is expressed in the SCN and eyes, it is still possible that mTIM has a clock-relevant function but that its function is distinct from that described for dTIM. It is also conceivable that an mTIM homolog other that the one characterized here might exist that interacts with the mPER proteins (Zylka, 1998b).

Individual variability in circadian locomotor activity has recently been discovered in the blind mole rat, Spalax ehrenbergi. An interesting association was found between different circadian types and two DNA fragments, 5.6 and 5.9 kb long, that contain the ACNGGN repeat sequence, homologous to a part of the period gene of Drosophila. Nine of 12 arrythmic animals showed the 5.6-kb band, while 13 of 17 circadian rhythmic animals had the 5.9-kb band. This repeat exists also in the brain RNA of the mole rat, apparently in higher quantities during the sleeping phase, suggesting that an unusual protein(s), composed of a poly-Thr-Gly segment, affect circadian rhythm (Ben-Shlomo, 1996).

The molecular components of mammalian circadian clocks are elusive. A human gene termed RIGUI (named after an ancient Chinese sundial) has been isolated that encodes a bHLH/PAS protein 44% homologous to Drosophila period. The highly conserved mouse homolog (m-rigui) is expressed in a circadian pattern in the suprachiasmatic nucleus (SCN), the master regulator of circadian clocks in mammals. Circadian expression in the SCN continues in constant darkness; a shift in the light/dark cycle evokes a proportional shift of m-rigui expression in the SCN. m-rigui transcripts also appear in a periodic pattern in Purkinje neurons, pars tuberalis, and retina, but with a timing of oscillation different from that seen in the SCN. Expression of m-rigui in the pars tuberalis is lacking in inbred mice strains that have a genetic defect for pineal melatonin biosynthesis. Sequence homology and circadian patterns of expression suggest that RIGUI is a mammalian ortholog of the Drosophila period gene, raising the possibility that a regulator of circadian clocks is conserved (Sun, 1997).

The pervasive role of circadian clocks in regulating physiology and behavior is widely recognized. Their adaptive value lies in their ability to be entrained by environmental cues such that the internal circadian phase is a reliable predictor of solar time. In mammals, both light and nonphotic behavioral cues can entrain the principal oscillator of the hypothalamic suprachiasmatic nuclei (SCN). However, although light can advance or delay the clock during circadian night, behavioral events trigger phase advances during the subjective day, when the clock is insensitive to light. The recent identification of Period (Per) genes in mammals, homologs of Drosophila period, which encodes a core element of the circadian clockwork in Drosophila, now provides the opportunity to explain circadian timing and entrainment at the molecular level. In mice, expression of mPer1 and mPer2 in the SCN is rhythmic and acutely up-regulated by light. The temporal relations between mRNA and protein cycles are consistent with a clock based on a transcriptional/translational feedback loop. Circadian oscillations of Per1 and Per2 in the SCN of the Syrian hamster are described, showing that PER1 protein and mRNA cycles again behave in a manner consistent with a negative-feedback oscillator. The highest level of expression for both genes is in the SCN, where there is a strong cycle of expression, with peak levels in the early light phase (ZT 4), and a nadir in subjective night (ZT 16-ZT 20). Levels of expression start to rise again by the end of circadian night). As a result, there is a pronounced daily cycle in the relative intensity of the hybridization signal for both genes in the SCN. The highly significant interaction between time and gene arises from differential phasing of two rhythms, with mPer1 rising earlier and declining sooner than mPer2. The phase delay of mPer2 relative to mPer1 is approximately 3 to 4 hr. The pattern of expression of these genes has been studied in the SCN of hamsters subjected to a much-studied and potent nonphotic resetting cue, namely confinement to a running wheel that generally elicits considerable activity and arousal, driving the clock to a new phase. Nonphotic resetting has the opposite effect as light: the acute down-regulation of these genes. Their sensitivity to nonphotic resetting cues supports the proposed roles of these genes as core elements of the circadian oscillator. Moreover, this study provides an explanation at the molecular level for the contrasting but convergent effects of photic and nonphotic cues on the clock (Maywood, 1999).

A clear distinction has been established between the signaling pathways that mediate photic and nonphotic resetting. Photic induction of mPer in the SCN is probably mediated by glutamatergic retinal afferents, acting through a signaling cascade based on increased intracellular calcium and activation of the transcription factors CREB and ERK. In contrast, nonphotic resetting, through confinement to a novel wheel or scheduled arousal, requires neuropeptide Y (NPY)-ergic innervation of the SCN; there is a strong prediction from the current work that resetting by local infusion of NPY will be accompanied by a rapid suppression of the expression of mPer1 and mPer2 in the SCN. The potential for negative regulation of the transcriptional apparatus of the SCN by nonphotic cues has been demonstrated by the reported suppression of cFOS immunoreactivity in the SCN of hamsters subjected to a novel wheel, although the role played by this and other immediate-early gene products in the regulation of mPer is not yet known. The current identification of mPer genes as targets for contrasting resetting cues (light and behavioral inputs) suggests that novel therapeutic agents for manipulation of clock-related disorders could be identified by examining their actions on the expression of these genes in the SCN. Furthermore, the most appropriate time for the use of such agents, i.e., their phase dependence, could be predicted from the regulation of their molecular targets, either positive or negative (Maywood, 1999).

mPer1, a mouse gene, is a homolog of the Drosophila clock gene period and has been shown to be closely associated with the light-induced resetting of a mammalian circadian clock. To investigate whether the rapid induction of mPer1 after light exposure is necessary for light-induced phase shifting, an antisense phosphotioate oligonucleotide (ODN) to mPer1 mRNA was injected into the cerebral ventricle. Light-induced phase delay of locomotor activity at CT16 is significantly inhibited when the mice are pretreated with mPer1 antisense ODN 1 hr before light exposure. In addition, glutamate-induced phase delay of the suprachiasmatic nucleus (SCN) firing rhythm is attenuated by pretreatment with mPer1 antisense ODN. The present results demonstrate that induction of mPer1 mRNA is required for light- or glutamate-induced phase shifting, suggesting that the acute induction of mPer1 mRNA in the SCN after light exposure is involved in light-induced phase shifting of the overt rhythm (Akiyama, 1999).

Although the suprachiasmatic nucleus (SCN) is the major pacemaker in mammals, the peripheral cells or immortalized cells also contain a circadian clock. The SCN and the periphery may use different entraining signals -- light and some humoral factors, respectively. Induction of the circadian oscillation of gene expression is triggered by TPA treatment of NIH-3T3 fibroblasts. This induction is inhibited by a MEK inhibitor, and prolonged activation of the MAPK cascade is sufficient to trigger circadian gene expression. Therefore, prolonged activation of MAPK by entraining cues may be involved in the resetting of the circadian clock (Akashi, 2000).

Light-induced entrainment of the circadian clock is accompanied by the induction of some immediate-early genes in the SCN, and the serum shock, which triggers the induction of the circadian gene expression in cultured cells, also results in a transient and immediate induction of some genes such as mPer1. The acute induction of mPer1 mRNA in the SCN after light exposure is thought to be involved in light-induced phase shifting of the overt rhythm. Stimuli were sought that can induce the transient expression of mPer1 in mouse fibroblast NIH-3T3 cells and it was found that TPA treatment as well as a serum shock is able to induce the transient and strong expression of mPer1. The mRNA expression levels of mPer1 and mPer2 and albumin site D-binding protein (DBP), a clock-related gene encoding transcription factor, were monitored during 2 days. After the transient exposure to 50% serum, expression levels of all the three mRNAs oscillate with an approximate period length of 24 hr in confluently grown NIH-3T3 cells in the absence of serum. Thus, the serum shock is able to trigger the induction of a circadian oscillation of expression of clock and clock-related genes in NIH-3T3 cells as well as in Rat-1 cells. Remarkably, TPA treatment without serum also triggers the induction of a circadian oscillation of expression of the three genes, mPer1, mPer2, and DBP, with essentially the same period length as seen in serum-shocked cells. The TPA treatment is as effective as the serum shock in triggering the induction of circadian gene expression. Pretreatment with a specific inhibitor of protein kinase C (PKC) abolishes the TPA-induced circadian oscillation of gene expressions, confirming that TPA exerts its effect through activation of PKC. In contrast, the addition of the PKC inhibitor after TPA treatment failed to inhibit the triggering of the induction of circadian gene expression, suggesting that the inhibitor does not have a toxic effect. These results suggest that PKC activation is able to entrain the circadian rhythm of the gene expression (Akashi, 2000).

These results indicate that prolonged activation of the classic MAPK cascade (MEK/ERK) is able to induce immediate expression of mPer1 and trigger the induction of the circadian oscillation of expression of clock and clock-related genes in mammalian cultured cells and therefore suggest that the MAPK cascade has a key role in entrainment of the circadian rhythm in cultured cells. Previous studies have suggested that not only the SCN but also the periphery has a circadian clock, and light and some humoral factor(s) may act as an entraining cue in the SCN and the periphery, respectively; transcriptional activation is an essential event linking the cue and the circadian entrainment as well. In the SCN, light induces both ERK activation and immediate-early gene expression, which in general is mediated by the ERK pathway. It has also been reported that PKC activation in the SCN may have a role in rodent circadian rhythm and that treatment of the SCN with NGF induces the phase shift of circadian rhythm. Taken together, these results suggest that the MAPK cascade may function as a key mediator in common with the signal transduction pathways for entrainment of circadian rhythm in the SCN, the periphery, and immortalized cultured cells and that circadian entrainment by light and humoral factors may employ similar signal transduction mechanisms. These results also suggest that an unidentified humoral factor(s) that functions as an entraining cue in the periphery may induce the prolonged activation of the MAPK cascade in peripheral cells (Akashi, 2000 and references therein).

Nuclear entry of circadian oscillatory gene products is a key step for the generation of a 24-hr cycle of the biological clock. Nuclear import of clock proteins of the mammalian period gene family and the effect of serum shock, which induces a synchronous clock in cultured cells, have been examined. mCRY1 and mCRY2 have been shown to complex with PER proteins leading to nuclear import. Nuclear translocation of mPER1 and mPER2 (1) involves physical interactions with mPER3; (2) is accelerated by serum treatment, and (3) still occurs in mCry1/mCry2 double-deficient cells lacking a functional biological clock. Moreover, nuclear localization of endogenous mPER1 is observed in cultured mCry1/mCry2 double-deficient cells as well as in the liver and the suprachiasmatic nuclei (SCN) of mCry1/mCry2 double-mutant mice. This indicates that nuclear translocation of at least mPER1 also can occur under physiological conditions (i.e., in the intact mouse) in the absence of any CRY protein. The mPER3 amino acid sequence predicts the presence of a cytoplasmic localization domain (CLD) and a nuclear localization signal (NLS). Deletion analysis suggests that the interplay of the CLD and NLS proposed to regulate nuclear entry of PER in Drosophila is conserved in mammals, but with the novel twist that mPER3 can act as the dimerizing partner (Yagita, 2000).

It is speculated that activation of cell signaling pathways may ultimately lead to phosphorylation of mPER proteins, which in turn may facilitate the interaction and subsequent nuclear entry of mPER proteins. It is noteworthy that in Drosophila, phosphorylation of Per is essential for association with Tim and nuclear translocation of the Per/Tim complex. mPER3 contains sequences similar to the CLD and NLS of Drosophila Per. In mPER1 and mPER2, the CLD is conserved, but these proteins do not possess a typical single basic NLS, although mPER2 may have a bipartite NLS. There is both structural and functional conservation of the CLD. The dominance of the CLD over NLS activity in cellular localization of mPER3, as seen in Drosophila Per and Tim, has been confirmed. According to the fly model for CLD function, Per and TimM associate to mask the CLD of Per, allowing Tim to escort Per into the nucleus. By analogy, it is proposed that in mammals, heterodimerization of mPER1/mPER3 or mPER2/mPER3 masks the CLD of each partner, allowing the NLS of mPER3 to direct the heterodimers to the nucleus (Yagita, 2000).

The role of mammalian TIM in the nuclear transportation step of mPER proteins is unknown. In Drosophila, the subcellular localization of the Tim protein is regulated in a time-dependent fashion, but in mammalian SCN cells, mTIM is predominantly in the nucleus and does not exhibit circadian rhythmicity. Analogously, COS7 cells used in the present study constitutively express significant levels of endogenous mTim mRNA, even in the absence of a serum shock. mPER3-dependent nuclear translocation seems not to correlate with the level of mTim expression, because mTim mRNA levels are high in COS7 and wild-type embryonic fibroblasts, but low in rat-1 cells and mCry1/mCry2 double knockout embryonic fibroblasts (Yagita, 2000).

According to the basic model for the molecular mechanism of the circadian pacemaker, mPER proteins -- when in the nucleus -- repress CLOCK/BMAL-mediated transcriptional activation of various clock genes. Although mPER proteins inhibit transcription from mPer1- or vasopressin-promoter driven luciferase reporter constructs, repression is rarely complete, indicating that additional factors are required to completely suppress CLOCK/BMAL function. mCRY1 and mCRY2, members of the light-harvesting cryptochrome/photolyase protein family are indispensable components of the molecular oscillator because mice with inactivated mCry1 and mCry2 genes completely lack a biological clock. mCRY proteins strongly inhibit mPer1 and vasopressin promoter-driven luciferase expression in NIH-3T3 cells and they act as dimerization partners for translocation of mPER1, mPER2, and mPER3 into the nucleus. However, heterodimerization of mPER by itself was reported to affect cellular localization also. The current findings extend the significance of mPER-mPER interactions for nuclear translocation: mPER3 promotes the nuclear entry of mPER1 and mPER2. The pronounced nuclear localization of exogenous mPER1 in the mCRY-deficient MEFs after serum shock indicates that mPER3 can also accomplish nuclear entry of mPER1 without the help of mCRY proteins. Remarkably overexpression of mCRY1 or hCRY2 or both in mCry1/mCry2 double-mutant cells does not increase the mPER1-nuclear entry. In contrast, mPER3 transfection enhances mPER1 nuclear import in mCRY-deficient cells, whereas NLS-deleted mPER3 transfection markedly decreased the nuclear localization. Thus, it appears that there may be more than one route for mPER protein import into the nucleus. Moreover, immunocytochemical analysis using anti-mPER1-specific antisera reveals that endogenous mPER1 also localizes in the nucleus in cultured mCry1/mCry2 double-mutant cells, indicating that mCRY-independent nuclear translocation of exogenous mPER1 is not merely an artifact in cellular transfection studies. Importantly, mCRY-independent nuclear localization of endogenous mPER1 occurs in SCN and liver of mCry1/mCry2 double-mutant mice and thus is likely to be of physiological importance (Yagita, 2000).

The molecular oscillator that keeps circadian time is generated by a negative feedback loop. Nuclear entry of circadian regulatory proteins that inhibit transcription from E-box-containing promoters appears to be a critical component of this loop in both Drosophila and mammals. The Drosophila double-time gene product, a casein kinase Iepsilon (CKIepsilon) homolog, interacts with Drosophila Per and regulates circadian cycle length. Mammalian CKIepsilon binds to and phosphorylates the murine circadian regulator mPER1. Unlike both Drosophila Per and mPER2, mPER1 expressed alone in HEK 293 cells is predominantly a nuclear protein. Two distinct mechanisms appear to retard mPER1 nuclear entry: (1) coexpression of mPER2 leads to mPER1-mPER2 heterodimer formation and cytoplasmic colocalization; (2) coexpression of CKIepsilon leads to masking of the mPER1 nuclear localization signal and phosphorylation-dependent cytoplasmic retention of both proteins. CKIepsilon may regulate mammalian circadian rhythm by controlling the rate at which mPER1 enters the nucleus (Vielhaber, 2000).

An unexpected finding was that mPER1 expressed in HEK 293 cells is predominantly nuclear, while mPER2 is cytoplasmic. Coexpression of mPER1 with mPER2 or with active (but not inactive) CKIepsilon leads to accumulation of mPER1 in the cytoplasm rather than the nucleus. The CKI-dependent cytoplasmic localization requires a domain adjacent to the NLS in mPER1, implying that phosphorylation leads to a conformational change that masks the mPER1 NLS. These results suggest that both mPER2 and CKI can regulate mPER1 nuclear entry. The mechanism by which mPER2 keeps mPER1 in the cytoplasm appears to be distinct, and a study of the mPER1-mPER2 interaction is ongoing. Both mechanisms may allow for a delay in the negative regulation of circadian transcriptional activators such as CLOCK and BMAL1 (Vielhaber, 2000).

What mechanism finally allows nuclear entry of mPER protein complexes, leading to inhibition of CLOCK/BMAL1 activity? In Drosophila, heterodimerization of Per with tim allows nuclear import and subsequent inhibition of CLOCK/CYCLE transcription. However, there is no effect of mammalian TIM on mPER1 and mPER2 localization. In mammals, mCRY1 and mCRY2 have recently been shown to relocalize mPER1 and mPER2 proteins to the nucleus and efficiently repress transcription from E-box-containing promoters, although the mechanism by which mCRY proteins mediate this relocalization is not yet known. mCRY proteins may supply an NLS, although the data presented here raise the possibility that mCRY proteins could also allow unmasking of the mPER1 NLS by inhibition of CKI or recruitment of a specific phosphatase such as PP5 (Vielhaber, 2000).

Posttranslational regulation of clock proteins in mouse liver has been examined in vivo. The mouse PERIOD proteins (mPER1 and mPER2), CLOCK, and BMAL1 undergo robust circadian changes in phosphorylation. These proteins, the cryptochromes (mCRY1 and mCRY2), and casein kinase I epsilon (CKIepsilon) form multimeric complexes that are bound to DNA during negative transcriptional feedback. CLOCK:BMAL1 heterodimers remain bound to DNA over the circadian cycle. The temporal increase in mPER abundance controls the negative feedback interactions. Analysis of clock proteins in mCRY-deficient mice shows that the mCRYs are necessary for stabilizing phosphorylated mPER2 and for the nuclear accumulation of mPER1, mPER2, and CKIepsilon. in vivo evidence is provided that casein kinase I delta is a second clock relevant kinase (Lee, 2001).

These findings provide a novel mechanism by which the mPER proteins control the molecular clockwork; that is, the robust, high-amplitude oscillations in mPER protein abundance are necessary for perpetuating the circadian clock mechanism, since mPER proteins bring clock protein complexes into the nucleus at the proper time for negative transcriptional feedback. With rhythmic accumulation of either mPER1 or mPER2, the clock mechanism persists and drives circadian behavior for a period of time in constant conditions, as occurs when either mPer gene is targeted. Once in the nucleus, mPER2 appears to have the additional function of regulating Bmal1 transcription, leading to a more severe circadian phenotype with its disruption, compared with mPer1 disruption. When mPer1 and mPer2 are targeted together, the clock immediately ceases to function on placement in constant conditions, because the mPER rhythms are immediately disrupted (Lee, 2001).

Many aspects of mammalian physiology are driven through the coordinated action of internal circadian clocks. Clock speed (period) and phase (temporal alignment) are fundamental to an organism's ability to synchronize with its environment. In humans, lifestyles that disturb these clocks, such as shift work, increase the incidence of diseases such as cancer and diabetes. Casein kinases 1δ and ε are closely related clock components implicated in period determination. However, CK1δ is so dominant in this regard that it remains unclear what function CK1epsilon; normally serves. This study has revealed that CK1ε dictates how rapidly the clock is reset by environmental stimuli. Genetic disruption of CK1ε in mice enhances phase resetting of behavioral rhythms to acute light pulses and shifts in light cycle. This impact of CK1ε targeting is recapitulated in isolated brain suprachiasmatic nucleus and peripheral (lung) clocks during NMDA- or temperature-induced phase shift in association with altered PERIOD (PER) protein dynamics. Importantly, accelerated re-entrainment of the circadian system in vivo and in vitro can be achieved in wild-type animals through pharmacological inhibition of CK1ε. These studies therefore reveal a role for CK1ε in stabilizing the circadian clock against phase shift and highlight it as a novel target for minimizing physiological disturbance in shift workers (Pilorz, 2014).

The suprachiasmatic nucleus (SCN) of the anterior hypothalamus contains a major circadian pacemaker that imposes or entrains rhythmicity on other structures by generating a circadian pattern in electrical activity. The identification of 'clock genes' within the SCN and the ability to dynamically measure their rhythmicity by using transgenic animals open up new opportunities to study the relationship between molecular rhythmicity and other well-documented rhythms within the SCN. This study investigates SCN circadian rhythms in Per1-luc bioluminescence, electrical activity in vitro and in vivo, as well as the behavioral activity of rats exposed to a 6-hr advance in the light-dark cycle followed by constant darkness. The data indicate large and persisting phase advances in Per1-luc bioluminescence rhythmicity, transient phase advances in SCN electrical activity in vitro, and an absence of phase advances in SCN behavioral or electrical activity measured in vivo. Surprisingly, the in vitro phase-advanced electrical rhythm returns to the phase measured in vivo when the SCN remains in situ. This study indicates that hierarchical levels of organization within the circadian timing system influence SCN output and suggests a strong and unforeseen role of extra-SCN areas in regulating pacemaker function (Vansteense, 2003).

Taken together, the results lead to the following hypothesis. The phase advance in the light-dark schedule leads to a nearly complete phase advance of the Per1-luc bioluminescence rhythm and a transient advance in the SCN pacemaker mechanism, controlling electrical activity. Extra-SCN oscillators are not phase advanced by the shifted light-dark cycle and influence SCN electrical activity. Eventually, the extra-SCN oscillators are effective in entraining the SCN pacemaker to their phase. This is a novel hypothesis in that it postulates a powerful role for non-SCN regions in phase control of the SCN and has important implications for understanding problems associated with shift work and transmeridian air travel (Vansteense, 2003).

The circadian clock in the suprachiasmatic nucleus of the hypothalamus (SCN) contains multiple autonomous single-cell circadian oscillators and their basic intracellular oscillatory mechanism is beginning to be identified. Less well understood is how individual SCN cells create an integrated tissue pacemaker that produces a coherent read-out to the rest of the organism. Intercellular coupling mechanisms must coordinate individual cellular periods to generate the averaged, genotype-specific circadian period of whole animals. To noninvasively dissociate this circadian oscillatory network in vivo, an experimental paradigm has been developed that exposes animals to exotic light-dark (LD) cycles with periods close to the limits of circadian entrainment. If individual oscillators with different periods are loosely coupled within the network, perhaps some of them would be synchronized to the external cycle while others remain unentrained. In fact, rats exposed to an artificially short 22 hr LD cycle express two stable circadian motor activity rhythms with different period lengths in individual animals. Analysis of SCN gene expression under such conditions suggests that these two motor activity rhythms reflect the separate activities of two oscillators in the anatomically defined ventrolateral and dorsomedial SCN subdivisions. Under forced desynchronization, two regions within the SCN oscillate out of phase, with mRNAs characteristic of day (Per1) and night (Bmal1) simultaneously expressed in ventrolateral and dorsomedial subdivisions (data qualitatively similar to Per1 were obtained for the distribution of Per2). Day and night for the T_22h rhythm correspond to cycling gene expression ventrolaterally, whereas day and night for the tau>24h rhythm correspond to expression dorsomedially. This expermental model provides a unique opportunity to functionally dissect the overall output organization of the SCN in intact animals. The results suggest that either dorsomedial or ventrolateral SCN oscillators are capable of driving a motor activity rhythm. This 'forced desychronization' protocol has allowed the first stable separation of these two regional oscillators in vivo, correlating their activities to distinct behavioral outputs, and providing a powerful approach for understanding SCN tissue organization and signaling mechanisms in behaving animals (de la Iglesia, 2004).

The mammalian retina contains an endogenous circadian pacemaker that broadly regulates retinal physiology and function, yet the cellular origin and organization of the mammalian retinal circadian clock remains unclear. Circadian clock neurons generate daily rhythms via cell-autonomous autoregulatory clock gene networks. Thus, to localize circadian clock neurons within the mammalian retina, the cell type-specific expression of six core circadian clock genes was examined in individually identified mouse retinal neurons, and the clock gene expression rhythms in retinal degeneration (rd) mouse retinas were characterized. Individual photoreceptors, horizontal, bipolar, dopaminergic (DA) amacrines, catecholaminergic (CA) amacrines, and ganglion neurons were identified either by morphology or by a tyrosine hydroxylase (TH) promoter-driven red fluorescent protein (RFP) fluorescent reporter. Cells were collected, and their transcriptomes were subjected to multiplex single-cell RT-PCR for the core clock genes Period (Per) 1 and 2, Cryptochrome (Cry) 1 and 2, Clock, and Bmal1. Individual horizontal, bipolar, DA (dopaminergic), CA, and ganglion neurons, but not photoreceptors, were found to coordinately express all six core clock genes, with the lowest proportion of putative clock cells in photoreceptors (0%) and the highest proportion in DA neurons (30%). In addition, clock gene rhythms were found to persist for >25 days in isolated, cultured rd mouse retinas in which photoreceptors had degenerated. These results indicate that multiple types of retinal neurons are potential circadian clock neurons that express key elements of the circadian autoregulatory gene network and that the inner nuclear and ganglion cell layers of the mammalian retina contain functionally autonomous circadian clocks (Ruan, 2006).

Three mammalian Period (Per) genes, termed Per1, Per2, and Per3, have been identified as structural homologues of the Drosophila circadian clock gene, period. The three Per genes are rhythmically expressed in the suprachiasmatic nucleus (SCN), the central circadian pacemaker in mammals. The phases of peak mRNA levels for the three Per genes in the SCN are slightly different. Light sequentially induces the transcripts of Per1 and Per2 but not of Per3 in mice. These data and others suggest that each Per gene has a different but partially redundant function in mammals. To elucidate the function of Per1 in the circadian system in vivo, two transgenic rat lines were generated in which the mouse Per1 (mPer1) transcript was constitutively expressed under the control of either the human elongation factor-1alpha (EF-1alpha) or the rat neuron-specific enolase (NSE) promoter. The transgenic rats exhibited an ~0.6–1.0-h longer circadian period than their wild-type siblings in both activity and body temperature rhythms. Entrainment in response to light cycles was dramatically impaired in the transgenic rats. Molecular analysis revealed that the amplitudes of oscillation in the rat Per1 (rPer1) and rat Per2 (rPer2) mRNAs were significantly attenuated in the SCN and eyes of the transgenic rats. These results indicate that either the level of Per1, which is raised by overexpression, or its rhythmic expression, which is damped or abolished in over expressing animals, is critical for normal entrainment of behavior and molecular oscillation of other clock genes (Numano, 2006).

The mammalian circadian timing system is composed of a central pacemaker in the suprachiasmatic nucleus of the brain that synchronizes countless subsidiary oscillators in peripheral tissues. The rhythm-generating mechanism is thought to rely on a feedback loop involving positively and negatively acting transcription factors. BMAL1 and CLOCK activate the expression of Period (Per) and Cryptochrome (Cry) genes, and once PER and CRY proteins accumulate to a critical level they form complexes with BMAL1-CLOCK heterodimers and thereby repress the transcription of their own genes. This study shows that SIRT1, an NAD(+)-dependent protein deacetylase, is required for high-magnitude circadian transcription of several core clock genes, including Bmal1, Rorgamma, Per2, and Cry1. SIRT1 binds CLOCK-BMAL1 in a circadian manner and promotes the deacetylation and degradation of PER2. Given the NAD(+) dependence of SIRT1 deacetylase activity, it is likely that SIRT1 connects cellular metabolism to the circadian core clockwork circuitry (Asher, 2008).

In the current model of the mammalian circadian clock, PERIOD (PER) represses the activity of the circadian transcription factors BMAL1 and CLOCK, either independently or together with CRYPTOCHROME (CRY). This study provides evidence that PER has an entirely different function from that reported previously, namely, that PER inhibits CRY-mediated transcriptional repression through interference with CRY recruitment into the BMAL1-CLOCK complex. This indirect positive function of PER is consistent with previous data from genetic analyses using Per-deficient or mutant mice. Overall, the results support the hypothesis that PER plays different roles in different circadian phases: an early phase in which it suppresses CRY activity, and a later phase in which it acts as a transcriptional repressor with CRY. This buffering effect of PER on CRY might help to prolong the period of rhythmic gene expression. Additional studies are required to carefully examine the promoter- and phase-specific roles of PER (Akashi, 2014).