Mammalian Period homologs

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 T22h 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).

SIRT1 regulates circadian clock gene expression through PER2 deacetylation

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).

A positive role for PERIOD in mammalian circadian gene expression

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).

Circadian clock-controlled regulation of cGMP-protein kinase G

The suprachiasmatic nucleus (SCN) circadian clock exhibits a recurrent series of dynamic cellular states, characterized by the ability of exogenous signals to activate defined kinases that alter clock time. To explore potential relationships between kinase activation by exogenous signals and endogenous control mechanisms, clock-controlled protein kinase G (PKG: see Drosophila Foraging) regulation in the mammalian SCN were examined. Signaling via the cGMP-PKG pathway is required for light- or glutamate (GLU)-induced phase advance in late night. Spontaneous cGMP-PKG activation occura at the end of subjective night in free-running SCN in vitro. Phasing of the SCN rhythm in vitro is delayed by approximately 3 hr after treatment with guanylyl cyclase (GC) inhibitors, PKG inhibition, or antisense oligodeoxynucleotide (alphaODN) specific for PKG, but not PKA inhibitor or mismatched ODN. This sensitivity to GC-PKG inhibition was limited to the same 2 hr time window demarcated by clock-controlled activation of cGMP-PKG. Inhibition of the cGMP-PKG pathway at this time caused delays in the phasing of four endogenous rhythms: wheel-running activity, neuronal activity, cGMP, and Per1. Timing of the cGMP-PKG-necessary window in both rat and mouse depends on clock phase, established by the antecedent light/dark cycle rather than solar time. Because behavioral, neurophysiological, biochemical, and molecular rhythms show the same temporal sensitivities and qualitative responses, it is predicted that clock-regulated GC-cGMP-PKG activation may provide a necessary cue as to clock state at the end of the nocturnal domain. Because sensitivity to phase advance by light-GLU-activated GC-cGMP-PKG occurs in juxtaposition, these signals may induce a premature shift to this PKG-necessary clock state (Tischkau, 2003).

Rhythmic U2af26 Alternative Splicing Controls PERIOD1 Stability and the Circadian Clock in Mice

The circadian clock drives daily rhythms in gene expression to control metabolism, behavior, and physiology; while the underlying transcriptional feedback loops are well defined, the impact of alternative splicing on circadian biology remains poorly understood. This paper describes a robust circadian and light-inducible splicing switch that changes the reading frame of the mouse mRNA encoding U2-auxiliary-factor 26 (U2AF26). This results in translation far into the 3' UTR, generating a C terminus with homology to the Drosophila clock regulator Timeless. This new U2AF26 variant destabilizes PERIOD1 protein, and U2AF26-deficient mice show nearly arrhythmic PERIOD1 protein levels and broad defects in circadian mRNA expression in peripheral clocks. At the behavioral level, these mice display increased phase advance adaptation following experimental jet lag. These data suggest light-induced U2af26 alternative splicing to be a buffering mechanism that limits PERIOD1 induction, thus stabilizing the circadian clock against abnormal changes in light:dark conditions (Preussner, 2004).

Mouse Period homolog: Effects of mutation

Circadian rhythms are driven by endogenous biological clocks that regulate many biochemical, physiological and behavioural processes in a wide range of life forms. In mammals, there is a master circadian clock in the suprachiasmatic nucleus of the anterior hypothalamus. Three putative mammalian homologs (mPer1, mPer2 and mPer3) of the Drosophila circadian clock gene period (per) have been identified. The mPer genes share a conserved PAS domain (a dimerization domain found in Per, Arnt and Sim) and show a circadian expression pattern in the suprachiasmatic nucleus. To assess the in vivo function of mPer2, a deletion mutation in the PAS domain of the mouse mPer2 gene was generated and characterized. Mice homozygous for this mutation display a shorter circadian period followed by a loss of circadian rhythmicity in constant darkness. The mutation also diminishes the oscillating expression of both mPer1 and mPer2 in the suprachiasmatic nucleus, indicating that mPer2 may regulate mPer1 in vivo. These data provide evidence that an mPer gene functions in the circadian clock, and define mPer2 as a component of the mammalian circadian oscillator (Zheng, 1999).

Mice carrying a null mutation in the Period 1 (mPer1) gene were generated using embryonic stem cell technology. Homozygous mPer1 mutants display a shorter circadian period with reduced precision and stability. Mice deficient in both mPer1 and mPer2 do not express circadian rhythms. While mPER2 regulates clock gene expression at the transcriptional level, mPER1 is dispensable for the rhythmic RNA expression of mPer1 and mPer2 and may instead regulate mPER2 at a posttranscriptional level. Studies of clock-controlled genes (CCGs) reveal a complex pattern of regulation by mPER1 and mPER2, suggesting independent controls by the two proteins over some output pathways. Genes encoding key enzymes in heme biosynthesis are under circadian control and are regulated by mPER1 and mPER2. Together, these studies show that mPER1 and mPER2 have distinct and complementary roles in the mouse clock mechanism (Zheng, 2001).

The observation that the genes encoding the rate-limiting enzymes for heme biosynthesis, mAlas1 and mAlas2, are under circadian control is of particular interest. Circadian expression of mAlas1 and mAlas2 is completely disrupted in the double mutants, indicating that mPER1 and mPER2 regulate the availability of heme. The body level of cellular heme is tightly controlled, and this is thought to be achieved by a balance between heme synthesis and catabolism, the latter by heme-oxygenase. Two models have been proposed for the regulation of heme biosynthesis. The first model is based on a negative feedback of heme on Alas1 message stability and on a posttranslational transport control. A second model is based on a negative feedback of heme on the transcriptional control of Alas1 expression. A model based on a transcriptional control of mAlas1 expression by the circadian clock is a plausible mechanism for regulating levels of ALAS activity, and thereby heme levels, in mice. The clock control of the availability of heme may have a wider implication for temporal control of the biochemical and physiological processes of an organism. It is well known that heme serves as a prosthetic moiety for many heme proteins that are involved in a vast array of biological functions. Among key proteins that contain heme are proteins involved in oxygen metabolism (myoglobin, hemoglobin, catalase, etc), electron transfer (cytochromes c and p450), and signaling (guanylyl cyclases, nitric oxide synthase). It is thus possible that temporal control of heme biosynthesis could be a basis for a wider range of cascades in physiological processes. An interesting question raised here is whether the heme itself is important in regulating the clock. An independent link of heme to circadian regulation made possible by the fact that some PAS proteins are heme binding proteins. Recently, three bacteria PAS proteins, AxPDEA1, Dos, and FixL, have been shown to be heme binding proteins. For at least one of these, FixL, the PAS domain (in addition to being required for protein to protein interactions), is also required for heme binding. PAS motifs from bacteria to mammals have highly conserved three-dimensional folds even though the amino acid sequence identity is low. Thus, it is possible that other PAS proteins, including those in mammals, are potential heme binding proteins. Indeed, the mouse NPAS2, a protein that is highly homologous to CLOCK, is a heme binding protein. Both NPAS2 and CLOCK can form heterodimer with BMAL1 protein to form an active transcription complex. The regulation of heme level via mPER regulation of mAlas1/2 expression may be part of an interface between the core clock mechanism and the cellular/subcellular environment. It can be envisaged that the availability of heme controls the function of heme binding PAS proteins like NPAS2, which in turn regulates their activity with their transcriptional partner protein BMAL1. The transcriptional activity of the BMAL1/NPAS2 or BMAL1/CLOCK would then regulate the expression of clock genes such as the mPers. The levels of mPER proteins in turn regulate heme biosynthesis via control of mAlas1/2 expression. Such a model would provide a plausible interactive regulation between the biochemical/physiological conditions and the clock mechanism (Zheng, 2001).

The role of mPer1 and mPer2 in regulating circadian rhythms was assessed by disrupting these genes. Mice homozygous for the targeted allele of either mPer1 or mPer2 have severely disrupted locomotor activity rhythms during extended exposure to constant darkness. Clock gene RNA rhythms are blunted in the suprachiasmatic nucleus of mPer2 mutant mice, but not in mPER1-deficient mice. Peak mPER and mCRY1 protein levels are reduced in both lines. Behavioral rhythms of mPer1/mPer3 and mPer2/mPer3 double-mutant mice resemble rhythms of mice with disruption of mPer1 or mPer2 alone, respectively, confirming the placement of mPer3 outside the core circadian clockwork. In contrast, mPer1/mPer2 double-mutant mice are immediately arrhythmic. Thus, mPER1 influences rhythmicity primarily through interaction with other clock proteins, while mPER2 positively regulates rhythmic gene expression, and there is partial compensation between products of these two genes (Bae, 2001).

To assess the impact of targeted disruption of mPer1 on molecular rhythms, patterns of gene expression in the SCN were examined on the first day in DD. Rhythmic expression of mPer2, mCry1, and Bmal1 RNAs is unaltered in the SCN of mPER1-deficient mice. In contrast to the lack of effect on SCN gene expression, clock protein rhythms in the SCN are markedly altered in mPER1-deficient mice. In wild-type mice, robust rhythms of nuclear mPER1, mPER2, and mCRY1 were detected on the first day in DD. mPER1 staining is absent in mPER1-deficient mice (Bae, 2001).

Gene expression rhythms are markedly altered in mPer2 mutant mice. In the SCN of mice homozygous for the mPer2 mutation, mPer1, mCry1 levels are rhythmic, while mPer2 levels are not. Bmal1 RNA levels vary significantly with time, but the data are not sinusoidal in shape, e.g., not rhythmic per se. There is a significant main effect of circadian time for each of the four genes examined, as well as a significant effect of genotype (mCry1, Bmal1) or a significant interaction (mPer1, mPer2). Post-hoc, pairwise comparisons revealed that mice homozygous for the mPer2 mutation have significantly depressed peak levels of mPer1, mPer2, mCry1, and Bmal1 gene expression in the SCN. These results are consistent with previous studies showing reduced levels of gene expression in the SCN of mPer2 mutant mice and further support a role for mPER2 as a positive regulator within the circadian feedback loop (Bae, 2001).

The core oscillator driving the circadian clock is located in the ventral part of the hypothalamus, the so called suprachiasmatic nuclei (SCN). At the molecular level, this oscillator is thought to be composed of interlocking autoregulatory feedback loops involving a set of clock genes. Among the components driving the mammalian circadian clock are the Period 1 and 2 (mPer1 and mPer2) and Cryptochrome 1 and 2 (mCry1 and mCry2) genes. A mutation in the mPer2 gene leads to a gradual loss of circadian rhythmicity in mice kept in constant darkness (DD). Inactivation of the mCry2 gene in mPer2 mutant mice restores circadian rhythmicity and normal clock gene expression patterns. Thus, mCry2 can act as a nonallelic suppressor of mPer2, which points to direct or indirect interactions of PER2 and CRY2 proteins. In marked contrast, inactivation of mCry1 in mPer2 mutant mice does not restore circadian rhythmicity but instead results in complete behavioral arrhythmicity in DD, indicating different effects of mCry1 and mCry2 in the clock mechanism (Oster, 2002).

The Period2 gene plays a key role in controlling circadian rhythm in mice. Mice deficient in the mPer2 gene are cancer prone. After gamma radiation, these mice show a marked increase in tumor development and reduced apoptosis in thymocytes. The core circadian genes are induced by gamma radiation in wild-type mice but not in mPer2 mutant mice. Temporal expression of genes involved in cell cycle regulation and tumor suppression, such as Cyclin D1, Cyclin A, Mdm-2, and Gadd45alpha, is deregulated in mPer2 mutant mice. In particular, the transcription of c-myc is controlled directly by circadian regulators and is deregulated in the mPer2 mutant. BMAL1/NPAS2 or BMAL1/CLOCK heterodimers likely repress transcription of c-myc through E box-mediated reactions in the P1 promoter, and mPer2 can suppress c-myc expression indirectly through stimulating Bmal1 transcription. Deregulation of Bmal1 in mPer2m/m cells, therefore, results in c-myc overexpression. These studies suggest that the mPer2 gene functions in tumor suppression by regulating DNA damage-responsive pathways (Fu, 2002).

Based on recent discoveries from c-myc studies and the results of this study, a model is proposed for the role of mPer2 in tumor suppression. In this model, the loss of mPer2 function results in dampened Bmal1 expression and decreased intracellular levels of BMAL1/NPAS2 or BMAL1/CLOCK heterodimers, leading to the derepression of c-myc throughout 24 hr L/D cycles. Overexpression of c-myc causes genomic DNA damage and eventually leads to hyperplasia and tumor development. Following gamma radiation, the loss of mPer2 function partially impairs p53-mediated apoptosis, leading to accumulation of damaged cells. However, the mPer2m/m cells, expressing c-myc at elevated levels, can still progress through cell cycle in the presence of genomic DNA damage, resulting in the high incidence of tumor development after gamma radiation (Fu, 2002).

The mPer1, mPer2, mCry1, and mCry2 genes play a central role in the molecular mechanism driving the central pacemaker of the mammalian circadian clock, located in the suprachiasmatic nuclei (SCN) of the hypothalamus. In vitro studies suggest a close interaction of all mPER and mCRY proteins. mPER and mCRY interactions in vivo were investigated by generating different combinations of mPer/mCry double-mutant mice. mCry2 acts as a nonallelic suppressor of mPer2 in the core clock mechanism. Focus was placed on the circadian phenotypes of mPer1/mCry double-mutant animals; a decay of the clock with age was found in mPer1-/- mCry2-/- mice at the behavioral and the molecular levels. These findings indicate that complexes consisting of different combinations of mPER and mCRY proteins are not redundant in vivo and have different potentials in transcriptional regulation in the system of autoregulatory feedback loops driving the circadian clock (Oster, 2003b).

Old mPer1-/- mCry2-/- mice synchronize poorly to the light dark cycle. Therefore, tests were performed to see whether CREB, an essential factor for numerous transcriptional processes, is activated by phosphorylation in response to a light pulse. CREB phosphorylation was only slightly lowered in young mPer1-/- mCry2-/- mice but was significantly impaired in old animals, indicating a defect in light signaling in the SCN of these mice. At the behavioral level, the phase shifts of only young mPer1-/- mCry2-/- mice could be measured, because old animals immediately became arrhythmic in DD. The young mPer1-/- mCry2-/- mice resemble mPer1-/- animals in that they are not able to advance clock phase, suggesting that this anomaly is due to the absence of mPer1 (Oster, 2003b).

The impaired light response of mPer1-/- mCry2-/- mice might be a consequence of a defect in transmitting light information from the eye to the SCN. To test this possibility, anatomical malformations in the retina were sought. Neither young nor old mPer1-/- mCry2-/- mice displayed overt abnormalities in retinal morphology. Comparable to the SCN, however, light-dependent phosphorylation of CREB at Ser 133 was affected in old mPer1-/- mCry2-/- mice. As a consequence, light perceived by the eye is probably not processed properly to induce cellular signaling. The reason for the impaired transmission of the light signal is most likely not a developmental defect, because young mPer1-/- mCry2-/- mice show phosphorylation of CREB at Ser 133. Therefore, the defect is probably of transcriptional or posttranscriptional nature. The lack of phosphorylation of CREB might lead to an altered expression of melanopsin in ganglion cells. These cells are probably responsible for resetting of the clock by light. Hence, a reduced expression of melanopsin would affect resetting. This is in line with the recent finding, that melanopsin-deficient mice display attenuated clock resetting in response to brief light pulses, similar to what is observed in mPer1-/- mCry2-/- mice. In old mPer1-/- mCry2-/- mice, this might even lead to the poor synchronization of these mice to the LD cycle. Future studies will reveal whether melanopsin expression in ganglion cells of the retina is affected in old mPer1-/- mCry2-/- mice (Oster, 2003b).

The mammalian clock component PERIOD2 coordinates circadian output by interaction with nuclear receptors

Mammalian circadian clocks provide a temporal framework to synchronize biological functions. To obtain robust rhythms with a periodicity of about a day, these clocks use molecular oscillators consisting of two interlocked feedback loops. The core loop generates rhythms by transcriptional repression via the Period (PER) and Cryptochrome (CRY) proteins, whereas the stabilizing loop establishes roughly antiphasic rhythms via nuclear receptors. Nuclear receptors also govern many pathways that affect metabolism and physiology. This study shows that the core loop component PER2 can coordinate circadian output with the circadian oscillator. PER2 interacts with nuclear receptors including PPARalpha and REV-ERBalpha and serves as a coregulator of nuclear receptor-mediated transcription. Consequently, PER2 is rhythmically bound at the promoters of nuclear receptor target genes in vivo. In this way, the circadian oscillator can modulate the expression of nuclear receptor target genes like Bmal1, Hnf1alpha, and Glucose-6-phosphatase. The concept that PER2 may propagate clock information to metabolic pathways via nuclear receptors adds an important facet to the clock-dependent regulation of biological networks (Schmutz, 2010).

Comparing PER1 and PER2 proteins, a motif in PER2 was identified that extends its regulatory capacity, most probably by interaction with nuclear receptors. Between the two PAS domains, which mediate the interaction with BMAL1 and CLOCK, a potential LXXLL motif was identified. Substitution of its leucines by alanines or its deletion converted the Bmal1 regulatory potential of PER2 into that of PER1. However, replacing the LXXLL motifs of PER1 with the ones of PER2 did not convert PER1 into an activator of the Bmal1 gene. Consequently, there may be further amino acids involved that mediate or prevent interactions necessary for the activation of the Bmal1 gene. Recently, a partial tertiary structure of PER2 was resolved containing both PAS domains. In this structure, the LCCLL motif is embedded in a flexible linker region emerging from the PAS-A domain. One can speculate that the region between the two PAS domains could trigger interaction of PER2 with either BMAL1 and CLOCK, or nuclear receptors. The comparable region of PER1 may not be flexible enough or may not be accessible to allow both kinds of interactions. As a result, PER2 interacts much more strongly than PER1 with a selection of nuclear receptors (Schmutz, 2010).

Even though LXXLL-like motifs are important for interaction of nuclear receptors with PER2, this kind of interaction may not follow classical coactivator/nuclear receptor interactions. REV-ERBalpha does not possess a C-terminal helix 12 necessary to stabilize interaction with a typical LXXLL motif. In spite of this, Lys 456 was identified as important. In many nuclear receptors, a comparable lysine directly interacts with the two neighboring leucine residues of the LXXLL motif or similar amino acids of the coregulator N-CoR1. A similar function of this particular residue can be envisioned for the interaction of REV-ERBalpha with PER2. Other mutations of REV-ERBalpha -- e.g., preventing the binding of the prosthetic heme group by a deletion of the last 28 amino acids (δC) or by a replacement of His 602 with phenylalanine, or preventing the binding of the corepressor N-CoR1 (six amino acids that directly contact N-CoR1 replaced by alanines) -- did not abolish the interaction with PER2 (Schmutz, 2010).

PER2 may function as a modulator of Bmal1 gene expression when complemented with nuclear receptors. Cotransfection of PER2 with PPARalpha and RXRalpha yielded a dose-dependent increase of Bmal1 activity, while cotransfection of REV-ERBalpha reduced the activation potential of PER2. Since REV-ERBalpha recruits also the corepressor N-CoR1 for its repressive function on the Bmal1 promoter, one could envisage a competition between PER2 and N-CoR1 for the same binding pocket in REV-ERBalpha. This competition might be influenced by heme, because the activity of the REV-ERBalpha/N-CoR1 complex seems to be modulated by the presence of this ligand. In contrast, the interaction of REV-ERBalpha and PER2 is resistant to the depletion or augmentation of the endogenous heme pool in NIH 3T3 cells. At the moment, however, it cannot be ruled out that in vivo binding of heme might have a regulatory influence on the strength of this interaction, allowing integration of the metabolic state via the interaction of PER2 with REV-ERBalpha (Schmutz, 2010).

The data suggest that PER2 can bind to many nuclear receptor target genes in vivo. Surprisingly, this binding correlated with the binding of several nuclear receptors and occurred in many different phases of the circadian cycle. It is tempting to speculate that post-translational modifications of PER2 or the nuclear receptors confer specificity to such interactions. Two nuclear receptors were found in the regulatory regions of the Bmal1 gene in the liver: REV-ERBalpha and PPARalpha. At the promoter region, the rhythmic binding of REV-ERBalpha is in line with the hypothesis that it is a negative regulator of the Bmal1 gene. At the upstream regulatory element, rhythmic binding of PPARalpha was found in the phase of transcriptional activation. However, evidence was found that PER2 contributes to the circadian regulation of this gene as well. PER2 bound to two distinct regions of the Bmal1 gene in two different phases. At the promoter, PER2 interacts with REV-ERBalpha in a phase that correlates with transcriptional repression and contributes ~10% to the overall repression. In contrast, at the upstream regulatory region, the binding of PER2 contributes ~25% to the overall transcriptional activity. Most probably, this effect is mediated via PPARalpha. The results show for the first time that PER2 binds at the Bmal1 regulatory region, and thereby has an influence on the magnitude and amplitude of Bmal1 expression (Schmutz, 2010).

From genetic analysis, it is concluded that PER2 may affect the regulation of the Hnf1α gene in the liver. It is speculated that this effect might be mediated via HNF4α, because PER2 can be immunoprecipitated with this nuclear receptor and both proteins are present at the same time at the Hnf1α promoter. HNF1α and HNF4α are both transcription factors at the crossroads of glucose homeostasis. Mutations in those genes lead to maturity-onset diabetes in the young (MODY) caused by an insulin secretion defect. Hence, Per2Brdm1 mutant animals may show alterations in glucose homeostasis, as observed previously for Bmal1 and Clock mutant mice (Schmutz, 2010).

PER3 polymorphism in humans predicts sleep structure and waking performance

Circadian rhythmicity and sleep homeostasis interact to regulate sleep-wake cycles, but the genetic basis of individual differences in sleep-wake regulation remains largely unknown. PERIOD genes are thought to contribute to individual differences in sleep timing by affecting circadian rhythmicity, but not sleep homeostasis. This study quantified the contribution of a variable-number tandem-repeat polymorphism in the coding region of the circadian clock gene PERIOD3 (PER3) to sleep-wake regulation in a prospective study, in which 24 healthy participants were selected only on the basis of their PER3 genotype. Homozygosity for the longer allele (PER35/5) had a considerable effect on sleep structure, including several markers of sleep homeostasis: slow-wave sleep (SWS) and electroencephalogram (EEG) slow-wave activity in non-rapid eye movement (non-REM) sleep and theta and alpha activity during wakefulness and REM sleep were all increased in PER35/5 compared to PER34/4 individuals. In addition, the decrement of cognitive performance in response to sleep loss was significantly greater in the PER35/5 individuals. By contrast, the circadian rhythms of melatonin, cortisol, and peripheral PER3 mRNA expression were not affected. The data show that this polymorphism in PER3 predicts individual differences in the sleep-loss-induced decrement in performance and that this differential susceptibility may be mediated by its effects on sleep homeostasis (Viola, 2007).

Role of 3'-untranslated region-mediated mRNA decay in circadian oscillations of mammalian Period mRNA

Daily oscillations in mRNA levels are a general feature of most clock genes. Although mRNA oscillations largely depend on transcriptional regulation, it has been suggested that post-transcriptional controls also contribute to mRNA oscillations in Drosophila. Currently, however, there is no direct evidence for post-transcriptional regulation of mammalian clock genes. To investigate the roles of post-transcriptional regulations, focus was placed on the 3'-untranslated region (3'-UTR) of mouse Period3 (mPer3) mRNA, one of the clock genes. Insertion of the entire mPer3 3'-UTR downstream of a reporter gene resulted in a dramatic decrease in mRNA stability. Deletion and point mutation analyses led to the identification of critical sequences responsible for mRNA decay. To explore the effects of the mPer3 3'-UTR-mediated mRNA decay on circadian oscillations, NIH3T3 stable cell lines were established that express luciferase mRNA with wild-type or mutant mPer3 3'-UTR. Interestingly, a stabilizing mutation of 3'-UTR induced a significant alteration in the oscillation profile of luciferase mRNA. Above all, the peak time, during which the mRNAs reached their highest levels, was significantly delayed (for 12 h). In addition, the luciferase mRNA level with mutant 3'-UTR began to increase earlier than that in the presence of wild-type 3'-UTR. Consequently, luciferase mRNA with mutant 3'-UTR displayed oscillation patterns with a prolonged rising phase. These results indicate that mPer3 3'-UTR-mediated mRNA decay plays an essential role in mRNA cycling and provide direct evidence for post-transcriptional control of circadian mRNA oscillations (Kwak, 2006: full text of article).

Signaling upstream of Period homologs: Clock and the photoperiod response

Period Evolutionary homologs part 3/3 | back to part 1/3

period: Biological Overview | Regulation | Targets of Activity and Post-transcriptional Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

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