period

EVOLUTIONARY HOMOLOGS (part 3/3)

Signaling upstream of Period homologs: Clock and the photoperiod response

Cryptochrome (Cry), a photoreceptor for the circadian clock in Drosophila, binds to the clock component TIM in a light-dependent fashion and blocks its function. In mammals, genetic evidence suggests a role for Crys within the biological clock, distinct from hypothetical photoreceptor functions. Mammalian CRY1 and CRY2 have been shown to act as light-independent inhibitors of CLOCK-BMAL1, the activator driving Per1 transcription. CRY1 or CRY2 (or both) show light-independent interactions with CLOCK and BMAL1, as well as with PER1, PER2, and TIM. No systematic or substantive differences have been observed in the ability of hCRY1 or hCRY2 to inhibit CLOCK-BMAL1 in light and dark conditions. Thus, mammalian CRY proteins act as light-independent components of the circadian clock and probably regulate Per1 transcriptional cycling by contacting both the activator and its feedback inhibitors. The light-independent role of mammalian Cry proteins in circadian clock negative feedback contrasts sharply with that of Drosophila Cry, which has been demonstrated to function directly as a photoreceptor that regulates the action of the Per-Tim complex. It is suggested that Drosophila Cry exemplifies the ancestral role of a photoreceptor acting as a light-dependent regulator of the circadian feedback loop, whereas mammalian Crys have preserved a role within the circadian feedback loop but shed their direct photoreceptor function. The possibility cannot be excluded that mammalian Cry proteins act as photoreceptors for other possible functions, circadian or otherwise, not detected in these assays (Griffin, 1999).

Clock (see Drosophila Clock) is a semidominant mutation found in mice. Mice carrying the Clock mutation exhibit abnormalities of circadian behavior, including lengthening of endogenous period and loss of rhythmicity. Clock segregates as a single autosomal locus and lengthens the period of the circadian rhythm by about 1 hour in heterozygotes. In homozygote mutants, period lengthens by about 4 hours upon initial transfer to constant darkness, after which these mice lose persistent circadian rhythms. To identify the gene affected by this mutation, a high-resolution genetic map (> 1800 meioses) was generated of the Clock locus. Clock is 0.7 cM distal of Kit on mouse chromosome 5. Mapping shows that Clock lies within the W19H deletion. Complementation analysis of different Clock and W19H compound genotypes indicates that the Clock mutation behaves as an antimorph. In an antimorph, the heterozygote (wild type/Clock) is more severe than the mutant allele plus a deletion (Clock/deletion) but less severe than the phenotype of Clock/Clock. The wild type/deletion heterozygote hemizygous phenotype is indistinguishable from the homozygous wild-type phenotype. This order of phenotypic severity is the defining characteristic of an antimorphic allele. That the Clock/deletion phenotype is significantly more severe than the Clock/wild type phenotype indicates that the wild-type allele is interacting with the Clock mutant allele to ameliorate the severity of the Clock mutant phenotype. This antimorphic behavior of Clock strongly argues that Clock defines a gene centrally involved in the mammalian circadian system (King, 1997a).

Positional cloning was used to identify the circadian Clock gene in mice. Clock is a large transcription unit with 24 exons spanning 100,000 bp of DNA from which transcript classes of 7.5 and 10 kb arise. Clock encodes a member of the bHLH-PAS family of transcription factors. Clock PAS A and PAS B motifs as well as Clock's bHLH domain most closely resemble mouse Neuronal PAS2. The Clock PAS motifs are only distantly related to those of Drosophila Period, which has no bHLH domain. The Clock PAS motifs bear a closer sequence resemblance to the PAS motifs of Drosophila Single minded. In the Clock mutant allele, an AT nucleotide transversion in a splice donor site causes exon skipping and deletion of 51 amino acids in the Clock protein. Clock is a unique mammalian gene with known circadian function and with features predicting DNA binding, protein dimerization, and activation domains. In addition to being expressed in the hypothalamus and the eye, Clock is expressed in brain, testis, ovary, liver, heart, lung and kidney. Clock represents the second example of a PAS domain-containing Clock protein (in addition to Drosophila Period), which suggests that this motif may define an evolutionarily conserved feature of the circadian clock mechanism (King, 1997b).

As a complementary approach to positional cloning, in vivo complementation with bacterial artificial chromosome (BAC) clones expressed in transgenic mice was used to identify the circadian Clock gene. A 140 kb BAC transgene completely rescues both the long period and the loss-of-rhythm phenotypes in Clock mutant mice. Analysis with overlapping BAC transgenes demonstrates that a large transcription unit spanning 100,000 base pairs is, in fact, the Clock gene; it encodes a novel basic-helix-loop-helix-PAS domain protein. Overexpression of the Clock transgene can shorten period length beyond the wild-type range, which provides additional evidence that Clock is an integral component of the circadian pacemaking system. Taken together, these results provide a proof of principle that "cloning by rescue" is an efficient and definitive method in mice (Antoch, 1997).

A brain pacemaker driving circadian rhythms in mammals resides in the suprachiasmatic nuclei (SCN) of the anterior hypothalamus. The SCN sense changes in ambient lighting through both direct and indirect retina-to-SCN neural pathways. In this way, light synchronizes (entrains) the SCN and its driven output rhythms to the 24 hr day. An important feature of SCN organization is that it is a multioscillatory system with the entire clockwork residing in single neurons. Individual neurons dissociated from rat suprachiasmatic nucleus have been shown to express independently phased circadian firing rhythms. Interactions among these "clock cells" in the whole SCN serve to synchronize individual circadian clocks to generate coordinated circadian outputs. These outputs ultimately control a vast array of circadian rhythms in physiology and behavior (Jin, 1999 and references).

The transcriptional regulation of the clock-controlled arginine vasopressin gene in the suprachiasmatic nuclei (SCN) has been studied. Vasopressin is synthesized and released in a circadian manner from neurons in the SCN. The SCN-derived peptide rhythm is driven by rhythmic transcription of the vasopressin gene. Peptide released from axons within the SCN acts locally through vasopressin-V1 receptors to modulate the amplitude of the SCN neuronal firing rate rhythm. Vasopressinergic efferent projections from the SCN also transmit circadian output signals to distant hypothalamic and extrahypothalamic regions. Vasopressin of SCN origin also appears to have a specific output function in driving rhythmic hypothalamo-pituitary-adrenal axis (HPA) activity. Vasopressin released from SCN terminals in the dorsomedial hypothalamus and subparaventricular zone has a pronounced inhibitory effect on the release of the adrenal hormone corticosterone. Vasopressin release might affect descending projections from brain stem and spinal cord, thereby decreasing splanchnic neural activity and corticosterone release. The vasopressin-mediated inhibition of corticosterone release is one aspect of a two-part regulatory mechanism controlling the corticosterone rhythm. The second mechanism involves a vasopressin-independent stimulatory input from the SCN to the HPA (Jin, 1999 and references).

A core clock mechanism in mouse SCN appears to involve a transcriptional feedback loop in which CLOCK and BMAL1 are positive regulators and three mPeriod (mPer) genes are involved in negative feedback. The RNA rhythm of each mPer gene is severely blunted in Clock/Clock mice. The vasopressin RNA rhythm is abolished in the SCN of Clock/Clock animals, leading to markedly decreased peptide levels. Luciferase reporter gene assays show that CLOCK-BMAL1 heterodimers act through an E box enhancer in the vasopressin gene to activate transcription; this activation can be inhibited by the mPER and mTIM proteins. Deletion of the protein dimerization PAS domain and cytoplasmic localization domain from mPER1 (amino acids 179-495) eliminates the inhibitory effect of mPER1 on CLOCK-BMAL1-mediated transcription. This finding suggests that mPER1-mediated inhibition is dependent on protein-protein interactions. These data indicate that the transcriptional machinery of the core clockwork directly regulates a clock-controlled output rhythm (Jin, 1999).

Signaling upstream of Period homologs: Cryptochrome, a protein that interacts with mammalian Period homologs

Two mouse cryptochrome genes, mCry1 and mCry2 (see Drosophila Cryptochrome), act in the negative limb of the clock feedback loop. In other words, mCRY1/mPER and mCRY2/mPER dimers inhibit CLOCK:BMAL1-mediated transcriptional activation, thus resulting in the negative regulation of PER and TIM in the nucleus. In cell lines, mPER proteins (alone or in combination) have modest effects on their cellular location and ability to inhibit CLOCK:BMAL1-mediated transcription. When mPER3 is coexpressed with either mPER1 or mPER2, mPER3 is dramatically redistributed from cytoplasm to become expressed in both cytoplasm and nucleus. mPER1 is more effective than mPER2 in promoting nuclear entry of mPER3; that is, nucleus-only location was found in 3× more cells with mPER1 cotransfections, as compared with mPER2. Despite trying all possible combinations of mPER proteins with mTIM, including adding all four proteins at once, a nucleus-only location of mPER1 or mPER2 could not be induced in greater than 30% of NIH3T3 cells. This differs dramatically from the in vivo situation in which both mPER1 and mPER2 are entirely nuclear in SCN cells when detectable. Thus, it would appear that mPER function cannot be fully reconstituted in NIH3T3 cells. Since mTim fails to drive mPER nuclear localization, it is concluded that the mTim function is not conserved between flies and mammals. This suggests that there are other clock-relevant factors important for the nuclear translocation of the mPER proteins (Kume, 1999).

This suggests cryptochrome might be involved in the negative limb of the feedback loop that results in the lowering of transcriptional levels of clock related genes. Indeed, mCry1 and mCry2 RNA levels are reduced in the central and peripheral clocks of Clock/Clock mutant mice. mCRY1 and mCRY2 are nuclear proteins that interact with each of the mPER proteins, translocate each mPER protein from cytoplasm to nucleus, and are rhythmically expressed in the suprachiasmatic circadian clock. Luciferase reporter gene assays show that mCRY1 or mCRY2 alone abrogates CLOCK:BMAL1-E box-mediated transcription. The mPER and mCRY proteins appear to inhibit the transcriptional complex differentially (Kume, 1999).

In addition to a potential direct inhibitory effect of the mCRY proteins on the CLOCK:BMAL1-E box complex, the cryptochromes could also inhibit transcription by directly interacting with the mPER proteins and translocating them to the nucleus for subsequent transcriptional effects. To evaluate the potential for protein-protein interactions between the mCRY and mPER families, coimmunoprecipitation using epitope-tagged proteins was carried out. COS7 cells cotransfected with expression plasmids encoding mCRY1-HA and either mPER1-V5, mPER2-V5, mPER3-V5, or mTIM-V5 expressed each V5-tagged protein prior to immunoprecipitation. Immunoprecipitation with the HA antibody and analysis of the immunoprecipitated material with anti-V5 antibodies indicates the presence of heterodimeric interactions between mCRY1 and each of the mPER and mTIM proteins. Coimmunoprecipitation experiments using mCRY2-HA instead of mCRY1-HA similarily show the presence of heterodimeric interactions between mCRY2 and each of the mPER and mTIM proteins. Having shown that mCRY:mPER heterodimers could exist, it was next determined whether such interactions translocate the mPER proteins to the nucleus. In marked contrast to the lack of effect of any pairwise combination of mPER:mPER or mPER:mTIM interactions to translocate mPER1 and mPER2 to the nucleus, each mCRY protein profoundly changes the location of all three mPER proteins in NIH3T3 and COS7 cells. This is most apparent for mPER1 and mPER2, which are almost entirely nuclear after cotransfection with either mCRY1 or mCRY2. Curiously, each mCRY protein changes mPER3 from mainly cytoplasm only (>80%) to both cytoplasm and nucleus (>70%) to a degree similar to that induced by cotransfection of mPER3 with mPER1. However, when mPER3 is cotransfected with mPER1 and either mCRY1 or mCRY2, each of the three protein combinations change mPER3's location from 13%-20% exclusively nuclear location to an exclusively nuclear location in a majority of cells (Kume, 1999).

These data indicate that the mCRY proteins can heterodimerize with the mPER proteins and mTIM and that mCRY:mPER interactions mimic the in vivo situation, that is, the almost complete translocation of mPER1 and mPER2 to the nucleus. Moreover, trimeric interactions among the mPER and mCRY proteins appear necessary for complete nuclear translocation of mPER3. The data also suggest that the nuclear translocation of the mPER proteins is dependent on mCRY1 and mCRY2. The mCRY proteins, however, appear to be able to translocate to the nucleus independent of the mPERs. Even with massive overexpression of mCRY proteins in cell culture, they are always greater than 90% nuclear. If a PER partner were required for CRY nuclear translocation, a high CRY:PER ratio should result in cytoplasmic trapping of CRY. This was not observed (Kume, 1999).

The discovery of the functions of mCRY1 and mCRY2 within the clock feedback loop provides a sharper view of the molecular working of the mammalian clock. The cloning of a family of three mPer genes over the past 2 years has added to understanding of the negative limb of a mammalian clock feedback loop. But close examination of these putative clock elements and mTim has shown that they alone cannot fully explain the negative limb of the feedback loop. It thus seemed likely that other factors were involved. mCRY1 and mCRY2 have been shown to be major players in the negative limb of the clock feedback loop. These data also explain the strong loss-of-function phenotype of mCry1^-/-mCry2^-/- mice (Kume, 1999).

The cell culture data show that the mCRY proteins function as dimeric and potentially trimeric partners for the mPER proteins and that these interactions lead to the nuclear translocation and/or retention of the mPER proteins. This is in marked contrast to the inability of mTIM to translocate the three mPER proteins to the nucleus in cell culture and the invariant nature of endogenous mTIM levels in the nuclei of SCN neurons; mTIM immunoreactivity is present in the nucleus of most SCN neurons at all times throughout the circadian cycle. Thus, the mCRY proteins appear to function as nuclear translocators of the mPERs. In addition, mCRY nuclear translocation does not appear to be dependent on mPER:mCRY interactions. This is different from the situation in the fly in which Per:Tim heterodimers appear essential for the translocation of both Per and Tim to the nucleus (Kume, 1999).

The role of mTIM in the mammalian clock remains enigmatic. Even though mTIM does not appear to be important for the nuclear translocation of the mPER proteins, mTIM is localized to the nucleus in vivo, and it does cause a modest inhibition of CLOCK:BMAL1- and MOP4:BMAL1-mediated transcription in cell culture. In addition, mCRY1 and mCRY2 each appear capable of forming heterodimeric complexes with mTIM. Once in the nucleus, mTIM could therefore still have a role in modulating negative feedback of the mPER and/or mCRY1 rhythms. Another observation from these studies is the finding that the mCry1 gene forms its own interacting loop within the collective mammalian clock feedback mechanism. Evidence for this contention is substantial. mCry1 RNA and protein levels exhibit a circadian rhythm in the SCN; the RNA rhythm is dependent on a functional CLOCK protein, and the mCry1 promoter region contains a functional CACGTG E box. In fact, it is entirely possible that the mCry1 rhythm is the dominant oscillation in the mammalian clock feedback loop. This might explain the dominant circadian function of the mCry1 gene over mCry2, whose RNA levels do not oscillate. One normal mCry1 allele sustains normal circadian rhythms in behavior, while one mCry2 allele leads to arrhythmicity with increasing time in constant darkness. It is not known precisely how the mPER and mCRY proteins inhibit CLOCK:BMAL1-mediated transcription, but the data suggest differential sites of action. In the fly, multimeric complexes involving Per, Tim, and Clock appear to be important. It is thus possible that the mPER proteins, mTIM, and the mCRY proteins are all complexed with CLOCK. In addition, mCRY1 and mCRY2 appear to be capable of inhibiting E box-mediated transcription independent of CLOCK. This suggests that the mammalian cryptochromes also interact directly with either BMAL1 or the E box itself. Indeed, mCRY1 can bind tightly to dsDNA Sepharose. Even though the major components of the loop have been identified, the way in which a 24 hr time constant is incorporated into the mammalian clock loop has not been elucidated. Based on studies in Drosophila, posttranslational processes such as phosphorylation, proteosomal proteolysis, and gated nuclear entry are likely to contribute to the time delay. It is not yet known which component(s) of the loop is affected by these processes (Kume, 1999 and references).

In summary, the data show that mCRY1 and mCRY2 are redundant but still essential components of the negative limb of the clock feedback loop. The redundant function of these proteins explains the maintenance of circadian rhythmicity when either gene is deleted and explains the strong arrhythmic phenotype of double knockout mice. The different direction of period change in mCry1^-/- versus mCry2^-/- mice may result from differing affinities of these proteins for the mPER proteins or other clock components, and/or different levels of protein expression. It is predicted that the SCN of mCry1^-/-mCry2^-/- animals will show disrupted mPer RNA and protein rhythms with the mPER proteins stuck in the cytoplasm and mPer RNA levels at constant high values because of the absence of negative feedback. Placing the mammalian cryptochromes in the negative limb of the clock feedback loop sets forth a number of new hypotheses that can now be tested (Kume, 1999 and references).

Mice lacking mCry1 and mCry2 are behaviorally arrhythmic. mCry2 is rhythmically expressed in the SCN in a manner similar to mCry1. Cyclic expression of the clock genes mPer1 and mPer2 (mammalian Period genes 1 and 2) in the suprachiasmatic nucleus and peripheral tissues is abolished and mPer1 and mPer2 mRNA levels are constitutively high. These findings indicate that the biological clock is eliminated in the absence of both mCRY1 and mCRY2 and support the idea that mammalian CRY proteins act in the negative limb of the circadian feedback loop. The high mRNA levels of mPer1 and mPer2 in mCry1/mCry2-deficient mice suggest that mCRY proteins negatively affect mPer expression. The mCry double-mutant mice retain the ability to have mPer1 and mPer2 expression induced by a brief light stimulus known to phase-shift the biological clock in wild-type animals. Thus, mCRY1 and mCRY2 are dispensable for light-induced phase shifting of the biological clock. The data suggest that photoreceptors other than mCRY proteins and rod/cone opsins [for example, mammalian homologs of the recently discovered fish VA-opsin or Xenopus laevis melanopsin] may be responsible for photic entrainment. Although mPer transcription repression by mCRY proteins (and thus their function in core oscillation) is light-independent, the possibliity that mCRY proteins may act as photoreceptor proteins cannot be completely ruled out: (1) it is possible that phase-shifting is mediated via more than one photoreceptor system, implying functional redundancy; (2) mCRY proteins may be involved in transmitting light inputs to the clock other than those required for phase shifting, such as changes in the length of the day and night, and information on dusk and dawn. Future experiments should shed light on the mysterious blue-light receptor properties of mCRY proteins (Okamura, 1999).

Cryptochromes regulate the circadian clock in animals and plants. Humans and mice have two cryptochrome (Cry) genes. Mice lacking the Cry2 gene have reduced sensitivity to acute light induction of the circadian gene mPer1 in the suprachiasmatic nucleus (SCN) and have an intrinsic period 1 hr longer than normal. In this study, Cry1-/- and Cry1-/-Cry2-/- mice were generated and their circadian clocks were analyzed at behavioral and molecular levels. Behaviorally, the Cry1-/- mice have a circadian period 1 hr shorter than wild type and the Cry1-/-Cry2-/- mice are arrhythmic in constant darkness (DD). Biochemically, acute light induction of mPer1 mRNA in the SCN is blunted in Cry1-/- and abolished in Cry1-/-Cry2-/- mice. In contrast, the acute light induction of mPer2 in the SCN was intact in Cry1-/- and Cry1-/-Cry2-/- animals. Importantly, in double mutants, mPer1 expression is constitutively elevated and no rhythmicity is detected in either 12-hr light/12-hr dark or DD, whereas mPer2 expression appears rhythmic in 12-hr light/12-hr dark, but nonrhythmic in DD with intermediate levels. These results demonstrate that Cry1 and Cry2 are required for the normal expression of circadian behavioral rhythms, as well as circadian rhythms of mPer1 and mPer2 in the SCN. The differential regulation of mPer1 and mPer2 by light in Cry double mutants reveals a surprising complexity in the role of cryptochromes in mammals (Vitaterna, 1999).

In the mouse, the core mechanism for the master circadian clock consists of interacting positive and negative transcription and translation feedback loops. Analysis of Clock/Clock mutant mice, homozygous Period2^Brdm1 mutants, and Cryptochrome-deficient mice reveals substantially altered Bmal1 rhythms, consistent with the dominant role of Period2 in the positive regulation of the Bmal1 loop. In vitro analysis of Cryptochrome inhibition of Clock:BMAL1 heterodimer-mediated transcription shows that the inhibition is through direct protein:protein interactions, independent of the Period and Timeless proteins. Period2 is a positive regulator of the Bmal1 loop, and Cryptochromes are the negative regulators of the Period and Cryptochrome cycles (Shearman, 2000).

Bmal1 mRNA rhythm has been documented in mouse SCN using in situ hybridization with an antisense riboprobe to the two major Bmal1 transcripts found in the SCN. Wild-type mice exhibit a robust circadian rhythm in Bmal1 RNA levels, with low levels from circadian time (CT) 6 to 9 and peak levels from CT 15 to 18. The phase of the Bmal1 rhythm is opposite that of the mPer1, mPer2, and mPer3 RNA rhythms. In addition to driving rhythmic transcription of the mPer and mCry genes, it seems possible that CLOCK:BMAL1 heterodimers might simultaneously negatively regulate Bmal1 gene expression, as proposed for Clk regulation in Drosophila. If CLOCK:BMAL1 heterodimers negatively regulate Bmal1 gene expression and if the mutant CLOCK protein is ineffective in this activity, then Bmal1 RNA levels should be elevated and less rhythmic in homozygous Clock mutant (Clk/Clk) mice. Compared with wild types, however, Clk/Clk animals express a severely dampened circadian rhythm of Bmal1 RNA levels in the SCN. Trough Bmal1 RNA levels do not differ between Clk/Clk mice and wild types. The peak level of the RNA rhythm in homozygous Clk mutant mice is only ~30% of the peak value in wild types (Shearman, 2000).

The temporal profile of Clk RNA levels in the SCN of Clk/Clk mutant animals was examined, because Clk RNA levels have been shown to be decreased in the eye and hypothalamus of Clk/Clk mutant mice. Clk RNA levels do not manifest a circadian oscillation in mouse SCN. Clk RNA levels in the SCN of Clk/Clk mutant mice are not significantly different from those in the SCN of wild-type animals. Thus, the Clk mutation appears to alter regulation of Bmal1 gene expression in SCN, but not the regulation of the Clk gene itself. Clk expression may be decreased in other hypothalamic regions (Shearman, 2000).

The low levels of Bmal1 RNA in the SCN of homozygous Clk mutant animals show that CLOCK is not required for the negative regulation of Bmal1. Instead, these data indicate that CLOCK is actually necessary for the positive regulation of Bmal1. The positive effect of CLOCK on Bmal1 levels is probably indirect and may occur through the mPER and/or mCRY proteins, which are expressed in the nucleus of SCN neurons at the appropriate circadian time to enhance Bmal1 gene expression. In addition, the mPer1, mPer2, mPer3, mCry1, and mCry2 RNA oscillations are all down-regulated in Clk/Clk mutant mice. Reduced levels of the protein products of one or more of these genes may lead to the reduced levels of Bmal1 in the mutant mice, through loss of a positive drive on Bmal1 transcription (Shearman, 2000).

Homozygous mPer2^Brdm1 mutant animals have depressed mPer1 and mPer2 RNA rhythms. The Bmal1 rhythm was examined in homozygous mPer2^Brdm1 mutants to determine whether the positive drive on the Bmal1 feedback loop might come from the mPER2 protein. The effects of this mutation on the mCry1 RNA rhythm were also examined. The temporal profiles of gene expression were analyzed at six time points over the first day in DD in homozygous mPer2^Brdm1 mutant mice and wild-type littermates. The Bmal1 RNA rhythm in the SCN of wild-type animals is substantially different from that of mutant mice. Trough RNA levels do not differ between wild-type and mutant animals, but the increase in Bmal1 RNA levels is advanced and truncated in the mutants, compared with the wild-type rhythm (Shearman, 2000).

The mCry1 RNA rhythm is also significantly altered. In the SCN of mPer2^Brdm1 mutant mice, the peak levels of the mCry1 RNA rhythm are suppressed by ~50%, as reported for mPer1 and mPer2 RNA rhythms in this mouse line. These data suggest that maintenance of a normal Bmal1 RNA rhythm is important for the positive transcriptional regulation of the mPer and mCry feedback loops. Thus, rhythmic Bmal1 RNA levels may drive rhythmic BMAL1 levels, which, in turn, regulate CLOCK:BMAL1-mediated transcriptional enhancement in the master clock. Indeed, mPer1, mPer2, and mCry1 RNA rhythms are all blunted in the SCN of mPer2^Brdm1 mutant mice, in which the Bmal1 rhythm is also blunted. In addition, the homozygous mPer2^Brdm1 mutation is associated with a shortened circadian period and ensuing arrhythmicity in DD (Shearman, 2000).

These data, along with the fact that Clk RNA levels are unaltered in the SCN of homozygous mPer2^Brdm1 mutants, also provide evidence that mPER2 is a positive regulator of the Bmal1 RNA rhythm. This effect may be unique to mPER2. For example, the diurnal oscillation in mPer2 RNA is not altered in the SCN of mPer1-deficient mice, and mPer1, mPer2, and Bmal1 RNA circadian rhythms are not altered in the SCN of mPer3-deficient mice. Moreover, circadian rhythms in behavior are sustained in mice deficient in either mPer1 or mPer3 (Shearman, 2000).

There are at least two ways that the mPer2^Brdm1 mutation could alter the positive drive of the clock feedback loops. The mutation could disrupt mPER:mCRY interactions important for the synchronous oscillations of their nuclear localization and/or alter the protein's ability to interact with other proteins (e.g., transcription factors). Experiments show that functional mPER2:mCRY interactions are not mediated through the PAS domain. Similarly, the PAS domain is not important for the mCRY-mediated nuclear translocation of mPER1 in COS-7 cells (Shearman, 2000).

Because the mPER:mCRY interactions necessary for nuclear transport occur outside the PAS region, the PAS domain of an mPER2:mCRY heterodimer might be free to bind to an activator (e.g., transcription factor) and shuttle it into the nucleus to activate Bmal1 transcription. Alternatively, once in the nucleus, mPER2:mCRY heterodimers or mPER2 monomers could coactivate Bmal1 transcription through a PAS-mediated interaction with a transcription factor. mPER2 itself does not have a DNA-binding motif (Shearman, 2000).

The tonic mid-to-high mPer1 and mPer2 RNA levels in mCry-deficient mice suggest that CLOCK:BMAL1 heterodimers might be constantly driving mPer1 and mPer2 gene expression in the absence of transcriptional inhibition by the mCRY proteins. To examine whether Bmal1 RNA levels would also be modestly elevated, Bmal1 RNA levels in the SCN of mCry-deficient mice were compared with those in the SCN of wild-type mice of the same genetic background at CT 6 and at CT 18. Clk RNA levels were also examined in these animals. In wild-type animals, the typical circadian variation in Bmal1 RNA levels is apparent with high levels at CT 18 and low levels at CT 6. In contrast, inmCry-deficient mice, Bmal1 RNA levels are low at both circadian times. It is suggested that Clk RNA levels do not differ as a function of circadian time or genotype (Shearman, 2000).

The unexpected low Bmal1 gene expression in the SCN of mCry-deficient mice suggests that the Bmal1 feedback loop is disrupted in the mutant animals, with a resultant nonfunctional circadian clock. Nevertheless, enough Bmal1 gene expression and protein synthesis occurs for heterodimerization with CLOCK so that, without the strong negative feedback normally exerted by the mCRY proteins, mPer1 and mPer2 gene expression is driven sufficiently by the heterodimer to give intermediate to high RNA values (depending on RNA stability) (Shearman, 2000).

The mid-to-high mPer1 and mPer2 RNA levels in the SCN of mCry-deficient mice and simultaneous low Bmal1 levels suggest that mPER1 and mPER2 proteins may not be exerting much positive or negative influence on the core feedback loops. To test this, it had to be determined whether mPER1 and mPER2 are tonically expressed in the nuclei of SCN cells in mCry-deficient mice, because nuclear location appears necessary for action on transcription. mPER1 immunoreactivity exhibits a robust rhythm of nuclear staining in the SCN of wild-type mice, with high values at CT 12 and significantly lower values at CT 24 (Shearman, 2000). In mCry-deficient mice, however, mPER1 immunoreactivity is detected in the nucleus of a similar number of SCN neurons at each of the two circadian times (CT 12 and CT 24), and the values at each time were at ~40% of those seen at peak (CT 12) in wild-type animals (Shearman, 2000).

The double mCry mutation also alters the subcellular distribution of mPER1 staining in the SCN. In wild-type mice, mPER1 staining viewed under contrast interference is nuclear with a very condensed immunoreaction and a clear nucleolus. The neuropil of the SCN in wild types is devoid of mPER1 immunoreactivity. In the SCN of mCry-deficient animals, mPER1 staining is nuclear, but the nuclear profiles are less well defined and less intensely stained: perinuclear, cytoplasmic immunoreaction can be observed. In addition, the neuropil staining for mPER1 is higher in mCry-deficient mice, although dendritic profiles are not discernible. In the same brains, the constitutive nuclear staining for mPER1 normally seen in the piriform cortex is not altered in mCry-deficient animals (Shearman, 2000).

mPER2 immunoreactivity also exhibits a robust rhythm of nuclear staining in the SCN of wild-type mice, with high values at CT 12 and significantly lower counts at CT 24. In contrast, the pattern of mPER2 immunoreactivity in the SCN of mCry-deficient mice is markedly altered, with few mPER2 immunoreactive cells in the SCN of mCry-deficient animals at either circadian time (CT 12 or CT 24) (Shearman, 2000).

In the wild-type mice, the mPER2 staining profiles are nuclear, with well-defined outlines and nucleoli devoid of reaction product. In the few mPER2 immunoreactive cells in the SCN of mCry-deficient mice, low-level mPER2 staining is observed in the nucleus, but the profiles are poorly defined and low-intensity perinuclear staining can also be observed. As for mPER1, genotype has no discernible effect on nuclear mPER2 immunoreactivity in the piriform cortex, although there is evidence of a low level of perinuclear immunoreactivity for mPER2 in piriform cortex of mCry-deficient mice. The marked reduction of mPER2 staining in the SCN of mCry-deficient animals suggests that the mCRY proteins are either directly or indirectly important for mPER2 stability, because mPer2 RNA levels are at tonic intermediate to high levels in mCry-deficient mice, similar to those found for mPer1 RNA levels. The low levels of mPER2 immunoreactivity in the SCN of mCry-deficient mice, in conjunction with tonically low Bmal1 RNA levels, are consistent with an important role of mPER2 in the positive regulation of the Bmal1 loop. Because mPER1 is present in SCN nuclei in mCry-deficient mice, yet Bmal1 RNA is low, mPER1 likely has little effect on the positive regulation of the Bmal1 feedback loop or negative regulation of the mPer1, mPer2, and mPer3 cycles (Shearman, 2000).

mPER1 and mPER2 can each enter the nucleus even in the absence of mCRY:mPER interactions. mPER1 is expressed in the nucleus of SCN neurons from mCry-deficient mice, and both mPER1 and mPER2 are constitutively expressed in the nucleus of cells in the piriform cortex of mCry-deficient animals. The phosphorylation state of mPER1 dictates its cellular location in the absence of mPER1:mCRY interactions, because its phosphorylation by casein kinase I epsilon leads to cytoplasmic retention in vitro. Thus, the nuclear location of both mPER1 and mPER2 in vivo may depend on several factors, including interactions with mCRY and other proteins and their phosphorylation (Shearman, 2000).

The intermediate to high levels of mPer1 and mPer2 gene expression throughout the circadian day in mCry-deficient mice are consistent with a prominent role of the mCRY proteins in negatively regulating CLOCK:BMAL1-mediated transcription. When cotransfected, mouse (m)CLOCK and syrian hamster (sh)BMAL1 heterodimers induce a large increase in transcriptional activity (1744-fold) that is reduced by >90% by mCRY1 or mCRY2. The mCLOCK: shBMAL1- and hMOP4:shBMAL1-induced transcription in S2 cells is dependent on an intact CACGTG E box, because neither heterodimer causes an increase in transcription when a mutated E box reporter is used in the transcriptional assay. Immunofluorescence of epitope-tagged mCRY1 or mCRY2 expressed in S2 cells shows that each is >90% nuclear in location, as in mammalian cells (Shearman, 2000).

These data indicate that mCRY1 and mCRY2 are nuclear proteins that can each inhibit mCLOCK:shBMAL1-induced transcription independent of the mPER and mTIM proteins and of each other. The results also show that the inhibitory effect is not mediated by the interaction of either mCRY1 or mCRY2 with the E box itself, because E box-mediated transcription is not blocked by the mCRY proteins when transcription is activated by dCLOCK:CYC heterodimers. It thus appears that the mCRY proteins inhibit mCLOCK: shBMAL1-mediated transcription by interacting with either or both of the transcription factors, because a similar inhibition is found with hMOP4:shBMAL1-induced transcription. Yeast two-hybrid assays have revealed strong interactions of each mCRY protein with mCLOCK and shBMAL1. Weaker interactions have been detected between each mCRY protein and hMOP4. This is further evidence of functionally relevant associations of each mCRY protein with each of the three transcription factors. Attempts were made to determine whether the mCRY-induced inhibition of transcription is through interaction with CLOCK and/or BMAL1. mCRY inhibits mCLOCK:shBMAL1-induced transcription through interaction with either mCLOCK alone or through an association with both mCLOCK and BMAL1 in a multiprotein complex (Shearman, 2000).

A working model of the SCN clockwork proposes three types of interacting molecular loops. The mCry genes comprise one loop, with true autoregulatory, negative feedback, in which the protein products feed back to turn off their own transcription. The second loop is that manifested by each of the mPer genes and some clock controlled output genes (CCGs: for example, vasopressin prepropressophysin). This loop type is driven by the same positive elements (CLOCK:BMAL1) as the mCry loop, but transcription is not turned off by the respective gene products. Instead, the mCRY protein acts as a negative regulator, leaving the protein products free for other actions. Thus, mPER2 positively drives transcription of the Bmal1 gene, mPER1 may function to stabilize protein components of the loop, and CCG products (which might include mPER3) function as output signals. The rhythmic regulation of Bmal1 comprises the third loop with rhythmicity controlled by the cycling presence and absence of a positive element dependent on mPER2. This positive feedback loop augments the positive regulation of the first two loops (Shearman, 2000).

This model of interacting loops proposes that at the start of the circadian day, mPer and mCry transcription are driven by accumulating CLOCK:BMAL1 heterodimers acting through E box enhancers. After a delay, the mPER and mCRY proteins are synchronously expressed in the nucleus where the mCRY proteins shut off CLOCK:BMAL1-mediated transcription by directly interacting with these transcription factors. At the same time that the mCRY proteins are inhibiting CLOCK:BMAL1-mediated transcription, mPER2 either shuttles a transcriptional activator into the nucleus or coactivates a transcriptional complex to enhance Bmal1 transcription. The importance of the Bmal1 RNA rhythm is to drive a BMAL1 rhythm after a 4- to 6-hour delay. This delay in the protein rhythm would provide increasingly available CLOCK:BMAL1 heterodimers at the appropriate circadian time to drive mPer/mCry transcription, thereby restarting the cycle. It is thus predicted that BMAL1 availability is rate limiting for heterodimer formation and critical for restarting the loops. Delineating factors that regulate clock protein stability and interactions (phosphorylation and proteolysis) are important next steps for defining how a 24-hour time constant is built into the clockwork (Shearman, 2000).

The core oscillator generating circadian rhythms in eukaryotes is composed of transcription-translation-based autoregulatory feedback loops in which clock gene products negatively affect their own expression. A key step in this mechanism involves the periodic nuclear accumulation of clock proteins following their mRNA rhythms after ~6 h delay. Nuclear accumulation of mPER2 is promoted by mCRY proteins. Using COS7 cells and mCry1/mCry2 double mutant mouse embryonic fibroblasts transiently expressing GFP-tagged (mutant) mPER2, it has been shown that the protein shuttles between nucleus and cytoplasm using functional nuclear localization and nuclear export sequences. Moreover, evidence is provided that mCRY proteins prevent ubiquitylation of mPER2 and subsequent degradation of the latter protein by the proteasome system. Interestingly, mPER2 in turn prevents ubiquitylation and degradation of mCRY proteins. On the basis of these data a model is proposed in which shuttling mPER2 is ubiquitylated and degraded by the proteasome unless it is retained in the nucleus by mCRY proteins (Yagita, 2002).

One of the key features of the self-sustaining circadian feedback loop is the phase delay of several hours between mRNA and protein peaks for genes involved in the circadian core oscillator. A mechanism of nuclear-cytoplasmic shuttling and ubiquitin-proteasome-dependent degradation obviously adds another level at which this delay can be modulated. Examination of the predicted amino acid sequence of other clock proteins reveals the presence of three putative NES domains in mPER1 (residues 138-149, 489-498 and 981-988) and mPER3 (residues 54-63, 399-408 and 913-920), all with conservation of the critical hydrophobic amino acid residues. In addition, human BMAL1 also contains two potential NES sequences (residues 105- 114 and 124- 134). It remains to be determined whether the mechanism of nuclear- cytoplasmic shuttling in conjunction with the ubiquitin-proteasome system is restricted to the mPER2 protein or whether it extends to other clock proteins. Similarly, it is not known whether shuttling of mPER2 (and perhaps other clock proteins) in combination with ubiquitin-proteasome-mediated degradation is clock regulated or whether it is a general mechanism involved in protein stabilization. Given the parallel with other systems the latter option is favored. Ubiquitylation and proteasomal degradation of clock proteins by itself is not a new finding. Light-dependent proteasomal degradation of Timeless has been described in Drosophila. However, this is the first example of the (light-independent) involvement of the ubiquitin-proteasome pathway in the core mechanism of the circadian oscillator (Yagita, 2002).

Receptor-mediated nucleocytoplasmic transport of clock proteins is an important, conserved element of the core mechanism for circadian rhythmicity. A systematic analysis of the nuclear export characteristics for the different murine period (mPER) and cryptochrome (mCRY) proteins using Xenopus oocytes as an experimental system demonstrates that all three mPER proteins, but neither mCRY1 nor mCRY2, are exported if injected individually. However, nuclear injection of heterodimeric complexes that contain combinations of mPER and mCRY proteins shows that mPER1 serves as an export adaptor for mCRY1 and mCRY2. Functional analysis of dominant-negative mPER1 variants designed either to sequester mPER3 to the cytoplasm or to inhibit nuclear export of mCRY1/2 in synchronized, stably transfected fibroblasts suggests that mPER1-mediated export of mCRY1/2 defines an important new element of the core clock machinery in vertebrates (Loop, 2005).

The molecular oscillator that drives circadian rhythmicity in mammals obtains its near 24-h periodicity from posttranslational regulation of clock proteins. Activity of the major clock kinase casein kinase I (CKI) epsilon is regulated by inhibitory autophosphorylation. Protein phosphatase (PP) 5 regulates the kinase activity of CKIepsilon. Cryptochrome regulates clock protein phosphorylation by modulating the effect of PP5 on CKIepsilon. Like CKIepsilon, PP5 is expressed both in the master circadian clock in the suprachiasmatic nuclei and in peripheral tissues independent of the clock. Expression of a dominant-negative PP5 mutant reduces Per phosphorylation by CKIepsilon in vivo, and down-regulation of PP5 significantly reduces the amplitude of circadian cycling in cultured human fibroblasts. Collectively, these findings indicate that PP5, CKIepsilon, and cryptochrome dynamically regulate the mammalian circadian clock (Partch, 2006).

Cryptochromes are composed of a core domain with structural similarity to photolyase and a distinguishing C-terminal extension. While plant and fly CRYs act as circadian photoreceptors, using the C terminus for light signaling, mammalian CRY1 and CRY2 are integral components of the circadian oscillator. However, the function of their C terminus remains to be resolved. The C-terminal extension of mCRY1 harbors a nuclear localization signal and a putative coiled-coil domain that drives nuclear localization via two independent mechanisms and shifts the equilibrium of shuttling mammalian CRY1 (mCRY1)/mammalian PER2 (mPER2) complexes towards the nucleus. Importantly, deletion of the complete C terminus prevents mCRY1 from repressing CLOCK/BMAL1-mediated transcription, whereas a plant photolyase gains this key clock function upon fusion to the last 100 amino acids of the mCRY1 core and its C terminus. Thus, the acquisition of different (species-specific) C termini during evolution not only functionally separated cryptochromes from photolyase but also caused diversity within the cryptochrome family (Chaves, 2006).

Interaction of circadian clock proteins CRY1 and PER2 is modulated by zinc binding and disulfide bond formation

Period (PER) proteins are essential components of the mammalian circadian clock. They form complexes with cryptochromes (CRY), which negatively regulate CLOCK/BMAL1-dependent transactivation of clock and clock-controlled genes. To define the roles of mammalian CRY/PER complexes in the circadian clock, the crystal structure of a complex comprising the photolyase homology region of mouse CRY1 (mCRY1) and a C-terminal mouse PER2 (mPER2) fragment was determined. mPER2 winds around the helical mCRY1 domain covering the binding sites of FBXL3 and CLOCK/BMAL1, but not the FAD binding pocket. The structure revealed an unexpected zinc ion in one interface, which stabilizes mCRY1-mPER2 interactions in vivo. Evidence is provided that mCRY1/mPER2 complex formation is modulated by an interplay of zinc binding and mCRY1 disulfide bond formation, which may be influenced by the redox state of the cell. These studies may allow for the development of circadian and metabolic modulators (Schmalen, 2014).

Signaling upstream of Period homologs: Hormonal and neurotransmitter regulation of the circadian clock

A circadian clock is located in the retinal photoreceptors of the African clawed frog Xenopus laevis. These photoreceptor clocks are thought to govern a wide variety of output rhythms, including melatonin release and gene expression. Both light and dopamine phase shift the retinal clock in a phase-dependent manner. Two homologs of the Drosophila period gene have been cloned in Xenopus, and one of these (xPer2) is acutely regulated by light. Light and dopamine induce xPer2 mRNA in a similar manner. In addition, the increase of xPer2 mRNA in response to light and dopamine is the same at all times of day tested. In contrast, xPer1 mRNA exhibits circadian oscillations but is relatively insensitive to phase-shifting treatments of light or dopamine. These data suggest that xPer2 functions as the molecular link between the light/dark cycle and the circadian clock (Steenhard, 2000).

The induction of xPer2 mRNA by dopamine in Xenopus retina is one of the first reports of a member of the per family being acutely responsive to a stimulus other than light. For many retinal processes, the effect of light is mimicked by dopamine. For example, light-evoked cone contraction is blocked by a D2-like dopamine antagonist, suggesting that dopamine release mediates light-evoked cone contraction. In contrast, light and dopamine act in parallel, convergent pathways to cause phase shifts in the melatonin rhythm. A D2-like antagonist is able to block the effects of exogenous dopamine but is ineffective in blocking phase shifts induced by light. D2-like receptor activation causes a decrease in cAMP levels. Increasing cAMP in Xenopus retina blocks phase shifting by dopamine but does not block phase shifting by light. Thus, light and dopamine cause phase shifts via different second messenger systems. Light and dopamine act in parallel to increase xPer2 mRNA. This may be the first point at which the light and dopamine pathways converge on the retinal clock (Steenhard, 2000 and references therein).

In mammals, the environmental light/dark cycle strongly synchronizes the circadian clock within the suprachiasmatic nuclei (SCN) to 24 hr. It is well known that not only photic but also nonphotic stimuli can entrain the SCN clock. Actually, many studies have shown that a daytime injection of 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH DPAT), a serotonin 1A/7 receptor agonist, as a nonphotic stimulus induces phase advances in hamster behavioral circadian rhythms in vivo, as well as the neuron activity rhythm of the SCN in vitro. Recent reports suggest that mammalian homologs of the Drosophila clock protein Period are involved in photic entrainment. Therefore, an examination was made to determine whether phase advances elicited by 8-OH DPAT are associated with a change of Period mRNA levels in the SCN. In this experiment, partial cDNAs were cloned encoding hamster Per1, Per2, and Per3 and both circadian oscillation and the light responsiveness of Period were observed. The inhibitory effect of 8-OH DPAT on hamster Per1 and Per2 mRNA levels in the SCN occurs only during the hamster's mid-subjective day, but not during the early subjective day or subjective night. The present findings demonstrate that the acute and circadian time-dependent reduction of Per1 and/or Per2 mRNA in the hamster SCN by 8-OH DPAT is strongly correlated with the phase resetting in response to 8-OH DPAT (Horikawa, 2000).

Many physiological and behavioral phenomena are controlled by an internal, self-sustaining oscillator with a periodicity of approximately 24 hr. In mammals, the principal oscillator resides in the suprachiasmatic nucleus (SCN). A light pulse during the subjective night causes a phase shift of the circadian rhythm via direct glutamatergic retinal afferents to the SCN. Along with the accepted theoretical models of the clock, it is suggested that behavioral resetting of mammals is completed within 2 hr; however, the molecular mechanism has not been elucidated. The real-time image of the transcription of the circadian-clock gene mPer1 is shown in the cultured SCN by using the transgenic mice that carry a luciferase reporter gene under the control of the mPer1 promoter. The real-time image demonstrates that the mPer1 promoter activity oscillates robustly in a circadian manner and that this promoter activity is reset rapidly (within 2-3 hr) when a phase shift occurs (Asai, 2001).

Signaling upstream of Period homologs: Phosphorylation

In mammals, the master circadian clock that drives many biochemical, physiological, and behavioral rhythms is located in the suprachiasmatic nuclei (SCN) of the hypothalamus. Generation and maintenance of circadian rhythmicity rely on complex interlocked transcriptional/translational feedback loops involving a set of clock genes. Among the molecular components driving the mammalian circadian clock are the Period 1 and 2 (mPer1 and mPer2) genes. Because the periodicity of the clock is not exactly 24 hr, it has to be adjusted periodically. The major stimulus for adjustment (resetting) of the clock is nocturnal light. It evokes activation of signaling pathways in the SCN that ultimately lead to expression of mPer1 and mPer2 genes conveying adjustment of the clock. Mice deficient in cGMP-dependent protein kinase II (cGKII, also known as PKGII), despite regular retinal function, are defective in resetting the circadian clock, as assessed by changes in the onset of wheel running activity after a light pulse. At the molecular level, light induction of mPer2 in the SCN is strongly reduced in the early period of the night, whereas mPer1 induction is elevated in cGKII-deficient mice. Additionally, light induction of cfos and light-dependent phosphorylation of CREB at serine 133 are not affected in these animals. It is concluded that cGKII plays a role in the clock-resetting mechanism. In particular, the ability to delay clock phase is affected in cGKII-deficient mice. It seems that the signaling pathway involving cGKII influences in an opposite manner the light-induced induction of mPer1 and mPer2 genes and thereby influences the direction of a phase shift of the circadian clock (Oster, 2003a).

Casein kinase 1 dynamics underlie substrate selectivity and the PER2 circadian phosphoswitch

Post-translational control of PERIOD stability by Casein Kinase 1delta/epsilon (CK1: Doubletime) plays a key regulatory role in metazoan circadian rhythms. Despite the deep evolutionary conservation of CK1 in eukaryotes, little is known about its regulation and the factors that influence substrate selectivity on functionally antagonistic sites in PERIOD that directly control circadian period. This study describes a molecular switch involving a highly conserved anion binding site in CK1. This switch controls conformation of the kinase activation loop and determines which sites on mammalian PER2 are preferentially phosphorylated, thereby directly regulating PER2 stability. Integrated experimental and computational studies shed light on the allosteric linkage between two anion binding sites that dynamically regulate kinase activity. Period-altering kinase mutations from humans to Drosophila differentially modulate this activation loop switch to elicit predictable changes in PER2 stability, providing a foundation to understand and further manipulate CK1 regulation of circadian rhythms (Philpott, 2020).

PERIOD1-associated proteins modulate the negative limb of the mammalian circadian oscillator

The clock proteins PERIOD1 (PER1) and PERIOD2 (PER2) play essential roles in a negative transcriptional feedback loop that generates circadian rhythms in mammalian cells. Two PER1-associated factors, NONO and WDR5, have been identified that modulate PER activity. The reduction of NONO expression by RNA interference (RNAi) attenuates circadian rhythms in mammalian cells, and fruit flies carrying a hypomorphic allele are nearly arrhythmic. WDR5, a subunit of histone methyltransferase complexes, augments PER-mediated transcriptional repression, and its reduction by RNAi diminishes circadian histone methylations at the promoter of a clock gene (Brown, 2005).

About 10% of all mammalian transcripts show daily oscillations of abundance. These rhythmic fluctuations are governed by a molecular circadian clock, whose function relies on two interconnected feedback loops of transcription. In the major negative feedback loop, transcription of the Period (Per1 and Per2) and Cryptochrome (Cry1 and Cry2) genes, and of Rev-Erbalpha, an orphan nuclear receptor gene, is activated by the transcription factors CLOCK and BMAL1 and repressed by the PERIOD (PER) and CRYPTOCHROME (CRY) proteins themselves. Although PERs interact with CRYs, the mechanism by which they repress CLOCK:BMAL1-mediated clock gene transcription remains poorly understood (Brown, 2005).

To elucidate the sizes of the PER1 and PER2 complexes involved in this process, nuclear extracts of mouse livers were fractionated by gel filtration chromatography. During the night, the two proteins formed similarly large complexes (>1 MD) whose abundance and size distribution changed during the day. Thus, PERs associate with other proteins that may play roles in the function of the circadian oscillator. To identify them, a Rat-1 fibroblast cell line was generated that expresses a PER1 protein containing a 6xHis tag and a V5 epitope tag at its C terminus. The expression of a His-V5-PER1 protein was three times higher than that of endogenous PER1 in unsynchronized cells and did not interfere with the circadian transcription of other clock or clock-controlled genes (Brown, 2005).

Nuclear extracts from His-V5-PER1 cells were harvested 3 hours after the induction of circadian rhythms by serum treatment. Because clock-controlled genes such as Dbp and Rev-erbalpha are repressed during this time period, PER-mediated transcriptional repression would likely be operative as well. After chromatography of this extract on nickel chelate resin, the eluted proteins (superose 6, and V5 antibody-Sepharose) were size-fractionated by gel electrophoresis and stained with colloidal Coomassie blue. Individual bands were excised, and the peptides they contained were identified by tandem mass spectrometry. In addition to peptides from CRY1 and CRY2, which are already known to be PER1-interacting proteins, peptides from two other factors, NONO and WDR5, were identified. NONO has been characterized previously as an RNA- and DNA-binding protein that could be involved in splicing, transcriptional repression, and RNA export; its Drosophila homolog, NonA, has been implicated in visual acuity and courtship behavior. WDR5 is a member of a histone methyltransferase complex and has been implicated in cell differentiation processes. RNA and proteins for both factors are expressed at constant levels throughout the day in a variety of tissues, including the suprachiasmatic nucleus, which is the site of the central circadian clock in the mammalian hypothalamus. However, the apparent sizes of the NONO protein complexes in liver nuclei varies during the day, and a size difference of these complexes was also observed between wild-type mice and mice lacking functional PER proteins (Brown, 2005).

NonA, the Drosophila homolog of NONO, plays an important physiological role, because flies harboring a P element-mediated deletion of this gene are extremely sick. However, because flies homozygous for the strongly hypomorphic nonA allele P14 are viable and fertile, it was possible to examine whether NonA also plays a role in the generation of circadian rhythms in Drosophila. nonA^P14/P14 flies are behaviorally nearly arrhythmic and hyperactive. In addition, the rhythmicity of mRNA from the clock gene timeless was greatly attenuated, indicating that the nonA^P14 mutation interferes with the basic function of the circadian oscillator (Brown, 2005).

The WDR5 protein has been identified as a member of a histone methyltransferase complex. Consistent with this function, histone methyltransferase activity can be precipitated from His-V5-PER1 fibroblast extracts with antibodies to V5 (anti-V5), anti-PER2, and anti-WDR5. It can also be enriched from evening but not morning nuclear extracts of mouse livers with anti-PER2. Because PER-regulated gene expression is low at night, it is likely that this methyltranferase activity has a repressive function. Such a repressive effect would be characteristic of histone H3 lysine 9 (H3K9) methylation, among other types. Nevertheless, WDR5 has been associated with a histone H3K4 methyltransferase containing a SET1 domain, which is mostly thought to be involved in the activation of transcription. Hence, both histone modifications were examined at a PER1-regulated circadian promoter in the presence and absence of WDR5 (Brown, 2005).

A lentivirally mediated system was used to generate a cell line that represses endogenous Wdr5 RNA and protein levels about five times in a doxycycline-inducible fashion. Circadian cycling in these cells was synchronized, and chromatin was harvested from cells grown in the presence or absence of doxycycline at the time of maximum and minimum Rev-erbalpha transcription. Methylation of histone H3K4 and histone H3K9 at the Rev-erbalpha promoter was examined by chromatin immunoprecipitation (ChIP). In uninduced cells expressing normal levels of WDR5, circadian methylation was observed in phase with Rev-erbalpha transcription at H3K4, and antiphase to it at H3K9. In doxycycline-treated cells, however, both rhythms were nearly abolished. Down-regulation of WDR5 expression produced only moderate changes in clock gene expression. PER-regulated genes such as Per2 and Rev-erbalpha were somewhat derepressed initially, and mRNA accumulation was then phase-delayed by about 2 hours. Similarly, when the same cells were analyzed with circadian luciferase reporter genes and real-time luminometry, the period length of cyclic gene expression was unchanged, suggesting that these two histone modifications are either compensatory or irrelevant for circadian clock function. Because overall bioluminescence from multiple reporters was also reduced, it is concluded that WDR5 reduction has a pleiotropic effect on cellular processes unrelated to circadian clock function. Therefore, although circadian histone methylation was drastically affected, a direct role for WDR5 in the circadian clock via loss-of-function experiments could not be verified. However, WDR5 can aid the PER-CRY complex in repressing CLOCK:BMAL1-mediated transcription, although this effect might be indirect (Brown, 2005).

This study identified two proteins, NONO and WDR5, that can associate with the mammalian PER1 protein. The data suggest that NONO probably operates antagonistically to PER proteins in mammalian cells, and that it is essential to normal circadian rhythmicity in mammals and in Drosophila. Mutations in the Drosophila homologs of PER1 and NONO were previously coidentified in a screen for courtship behavior defects. Overexpression experiments suggest that WDR5 assists PER function in mammalian cells. Its reduction affected two different antiphasic circadian histone modifications, H3K4 and H3K9, which are thought to have opposite effects, but WDR5 has only moderate consequences for circadian clock function. Hence, further studies of WDR5 and of histone posttranslational modifications at circadian promoters will be essential for understanding its contribution to clock function. It is suggested that the antagonistic activities of these proteins might help to render the circadian clock more resilient to noise -- caused by changes in temperature and nutrients, or by stochastic fluctuations in transcription rates -- that could affect the concentrations of critical clock components (Brown, 2005).

Transcriptional regulation of Period homologs

Transcript levels of DBP, a member of the PAR leucine zipper transcription factor family (see Drosophila Par-domain protein 1), exhibit a robust rhythm in suprachiasmatic nuclei, the mammalian circadian center. DBP is able to activate the promoter of a putative clock oscillating gene, mPer1, by directly binding to the mPer1 promoter. The mPer1 promoter is cooperatively activated by DBP and CLOCK-BMAL1. However, dbp transcription is activated by CLOCK-BMAL1 through E-boxes and inhibited by the mPER and mCRY proteins, as is the case for mPer1. Thus, a clock-controlled dbp gene may play an important role in central clock oscillation (Yamaguchi, 2000a).

The mPer1 gene is assumed to be a key molecule in the regulation and functioning of the mammalian circadian clock, which is based on the oscillation generated by a transcription-(post)translation feedback loop of a set of clock genes. Robust circadian oscillation and acute light-elicited induction of mPer1 mRNA expression have been observed in the suprachiasmatic nucleus (SCN), the mammalian circadian center. To investigate the mechanism underlying the complex regulation of mPer1 expression, the 5' upstream region of the mPer1 gene was isolated and characterized. Unexpectedly, two promoters, each followed by alternative first exons of mPer1, were identified. Consistent with the presence of multiple E-boxes in the promoters, exon-specific in situ hybridization of the SCN established that both promoters function in circadian oscillation and in light-induction of mPer1 expression. Transgenic mice carrying the 5' upstream region of the mPer1 gene fused to the luciferase gene have demonstrated that a DNA fragment carrying both promoter regions is sufficient to elicit striking circadian oscillation in the SCN and responsiveness to light. Moreover, luminescence in the SCN accurately mirrors the mPer1 transcriptional activity. These transgenic mice will be very useful for monitoring clock-specific mPer1 expression in intact organisms and to follow the circadian clock in real time (Yamaguchi, 2000b).

Light-dependent transcriptional regulation of clock genes is a crucial step in the entrainment of the circadian clock. E4bp4, a vertebrate ortholog of Drosophila Vrille, is a light-inducible gene in the chick pineal gland, and it encodes a bZIP protein that represses transcription of cPer2, a chick pineal clock gene. Prolonged light period-dependent accumulation of E4BP4 protein is temporally coordinated with a delay of the rising phase of cPer2 in the morning. E4BP4 is phosphorylated progressively and then disappears in parallel with induced cPer2 expression. Characterization of E4BP4 revealed Ser182, a phosphoacceptor site located at the amino-terminal border of the Ser/Thr cluster, which forms the phosphorylation motifs for casein kinase 1 (CK1 - Drosophila homolog Doubletime). This serine/threonine cluster is evolutionarily conserved from vertebrate E4BP4 to Drosophila Vrille. CK1 physically associates with E4BP4 and phosphorylates it. CK1-catalyzed phosphorylation of E4BP4 results in proteasomal proteolysis-dependent decrease of E4BP4 levels, while E4BP4 nuclear accumulation is attenuated by CK1 in a kinase activity-independent manner. CK1-mediated posttranslational regulation is accompanied by reduction of the transcriptional repression executed by E4BP4. These results not only demonstrate a phosphorylation-dependent regulatory mechanism for E4BP4 function but also highlight the role of CK1 as a negative regulator for E4BP4-mediated repression of cPer2 (Doi, 2004).

Circadian rhythms are generated by an extremely complicated transcription-translation feedback loop. To precisely analyze the molecular mechanisms of the circadian clock, it is critical to monitor multiple gene expressions and/or interactions with their transcription factors simultaneously. A novel reporter assay system, the tricolor reporter in vitro assay system, has been developed that consists of green- and red-emitting Phrixothrix luciferases as dual reporters and blue-emitting Renilla luciferase as internal control. This system has been successfully employed in analyzing the effects of clock gene products on the enhancer elements of Per1 and Bmal1 promoters. The results indicate that the orphan nuclear receptor RORalpha (see Drosophila Hormone receptor-like in 46) regulates bidirectionally Bmal1 (positively) and Per1 (negatively) transcriptions simultaneously (Nakajima, 2004).

Circadian clock genes are regulated through a transcriptional-translational feedback loop. Alterations of the chromatin structure by histone acetyltransferases and histone deacetylases (HDACs) are commonly implicated in the regulation of gene transcription. However, little is known about the transcriptional regulation of mammalian clock genes by chromatin modification. This study shows that the state of acetylated histones fluctuate in parallel with the rhythm of mouse Per1 (mPer1) or mPer2 expression in fibroblast cells and liver. Mouse CRY1 (mCRY1) represses transcription with HDACs and mSin3B, which is relieved by the HDAC inhibitor trichostatin A (TSA). In turn, TSA induces endogenous mPer1 expression as well as the acetylation of histones H3 and H4, which both interact with the mPer1 promoter region in fibroblast cells. Moreover, a light pulse stimulates rapid histone acetylation associated with the promoters of mPer1 or mPer2 in the suprachiasmatic nucleus (SCN) and the binding of phospho-CREB in the CRE of mPer1. TSA administration into the lateral ventricle induces mPer1 and mPer2 expression in the SCN. Taken together, these data indicate that the rhythmic transcription and light induction of clock genes are regulated by histone acetylation and deacetylation (Naruse, 2004).

The circadian clock within the cardiomyocyte is essential for responsiveness of the heart to fatty acids

Cells/organs must respond both rapidly and appropriately to increased fatty acid availability; failure to do so is associated with the development of skeletal muscle and hepatic insulin resistance, pancreatic beta-cell dysfunction, and myocardial contractile dysfunction. This study tested the hypothesis that the intrinsic circadian clock within the cardiomyocytes of the heart allows rapid and appropriate adaptation of this organ to fatty acids by investigating the following: (1) whether circadian rhythms in fatty acid responsiveness persist in isolated adult rat cardiomyocytes, and (2) whether manipulation of the circadian clock within the heart, either through light/dark (L/D) cycle or genetic disruptions, impairs responsiveness of the heart to fasting in vivo. Both the intramyocellular circadian clock and diurnal variations in fatty acid responsiveness observed in the intact rat heart in vivo persist in adult rat cardiomyocytes. Reversal of the 12-h/12-h L/D cycle is associated with a re-entrainment of the circadian clock within the rat heart, which requires 5-8 days for completion. Fasting rats result in the induction of fatty acid-responsive genes, an effect that is dramatically attenuated 2 days after L/D cycle reversal. Similarly, a targeted disruption of the circadian clock within the heart, through overexpression of a dominant negative Clock mutant, severely attenuates induction of myocardial fatty acid-responsive genes during fasting. These studies expose a causal relationship between the circadian clock within the cardiomyocyte with responsiveness of the heart to fatty acids and myocardial triglyceride metabolism (Durgan, 2006).

The circadian molecular clock regulates adult hippocampal neurogenesis by controlling the timing of cell-cycle entry and exit

The subgranular zone (SGZ) of the adult hippocampus contains a pool of quiescent neural progenitor cells (QNPs) that are capable of entering the cell cycle and producing newborn neurons. The mechanisms that control the timing and extent of adult neurogenesis are not well understood. This study shows that QNPs of the adult SGZ express molecular-clock components and proliferate in a rhythmic fashion. The clock proteins PERIOD2 and BMAL1 are critical for proper control of neurogenesis. The absence of PERIOD2 abolishes the gating of cell-cycle entrance of QNPs, whereas genetic ablation of bmal1 results in constitutively high levels of proliferation and delayed cell-cycle exit. Mathematical model simulations were used to show that these observations may arise from clock-driven expression of a cell-cycle inhibitor that targets the cyclin D/Cdk4-6 complex. These findings may have broad implications for the circadian clock in timing cell-cycle events of other stem cell populations throughout the body (Bouchard-Cannon, 2013).

The clock gene Period1 regulates innate routine behaviour in mice

Laboratory mice are well capable of performing innate routine behaviour programmes necessary for courtship, nest-building and exploratory activities although housed for decades in animal facilities. In mice, inactivation of the clock gene Period1 profoundly changes innate routine behaviour programmes like those necessary for courtship, nest building, exploration and learning. These results in wild-type and Period1 mutant mice, together with earlier findings on courtship behaviour in wild-type and period-mutant Drosophila melanogaster, suggest a conserved role of Period-genes on innate routine behaviour. Additionally, both per-mutant flies and Period1-mutant mice display spatial learning and memory deficits. The profound influence of Period1 on routine behaviour programmes in mice, including female partner choice, may be independent of its function as a circadian clock gene, since Period1-deficient mice display normal circadian behaviour (Bechstein, 2014)

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