Recently, a human cDNA clone with high sequence homology to the photolyase/blue-light photoreceptor family was identified. The putative protein encoded by this gene exhibits a strikingly high (48% identity) degree of homology to the Drosophila melanogaster (6-4) photolyase. A second human gene has been identified whose amino acid sequence displays 73% identity to the first one. The two genes have been named CRY1 and CRY2, respectively. The corresponding proteins hCRY1 and hCRY2 were purified and characterized as maltose-binding fusion proteins. Similar to other members of the photolyase/blue-light photoreceptor family, both proteins were found to contain FAD and a pterin cofactor. Like the plant blue-light photoreceptors, both hCRY1 and hCRY2 lack photolyase activity on the cyclobutane pyrimidine dimer and the (6-4) photoproduct. It is concluded that these newly discovered members of the photolyase/photoreceptor family are not photolyases and instead may function as blue-light photoreceptors in humans (Hsu, 1996).

Mouse photolyase-like genes, mCRY1 (mPHLL1) and mCRY2 (mPHLL2), which belong to the photolyase family including plant blue-light receptors, have been isolated and characterized. The mCRY1 and mCRY2 genes are located on chromosome 10C and 2E, respectively, and are expressed in all mouse organs examined. Antibodies specific against each gene product were raised using their C-terminal sequences, which differ completely between the genes. Immunofluorescent staining of cultured mouse cells reveals that mCRY1 is localized in mitochondria whereas mCRY2 was found mainly in the nucleus. Using green fluorescent protein fused peptides it has been demonstrated that the C-terminal region of the mouse CRY2 protein contains a unique nuclear localization signal, which is absent in the CRY1 protein. The N-terminal region of CRY1 contains the mitochondrial transport signal. Recombinant as well as native CRY1 proteins from mouse and human cells show a tight binding activity to DNA Sepharose, while CRY2 protein does not bind to DNA Sepharose at all under the same condition as CRY1. The different cellular localization and DNA binding properties of the mammalian photolyase homologs suggest that, despite the similarity in the sequence, the two proteins have distinct functions (Kobayashi, 1998).

In mammals the retina contains photoactive molecules responsible for both vision and circadian photoresponse systems. Opsins, which are located in rods and cones, are the pigments for vision but it is not known whether they play a role in circadian regulation. A subset of retinal ganglion cells with direct projections to the suprachiasmatic nucleus (SCN) are at the origin of the retinohypothalamic tract that transmits the light signal to the master circadian clock in the SCN. However, the ganglion cells are not known to contain rhodopsin or other opsins that may function as photoreceptors. Two blue-light photoreceptors, cryptochromes 1 and 2 (CRY1 and CRY2), recently discovered in mammals, are specifically expressed in the ganglion cell and inner nuclear layers of the mouse retina. In addition, CRY1 is expressed at high levels in the SCN and oscillates in this tissue in a circadian manner. These data, in conjunction with the established role of CRY2 in photoperiodism in plants, lead to a proposal that mammals have a vitamin A-based photopigment (opsin) for vision and a vitamin B2-based pigment (cryptochrome) for entrainment of the circadian clock (Miyamoto, 1998).

Molecular clocks do not oscillate with an exact 24-hour rhythmicity but are entrained to solar day/night rhythms by light. The mammalian proteins Cryl and Cry2, which are members of the family of plant blue-light receptors (cryptochromes) and photolyases, have been proposed as candidate light receptors for photoentrainment of the biological clock. Mice lacking the Cryl or Cry2 protein display accelerated and delayed free-running periodicity of locomotor activity, respectively. Strikingly, in the absence of both proteins, an instantaneous and complete loss of free-running rhythmicity is observed. This suggests that, in addition to a possible photoreceptor and antagonistic clock-adjusting function, both proteins are essential for the maintenance of circadian rhythmicity (van der Horst, 1999).

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).

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).

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).

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).

Two mouse cryptochrome genes, mCry1 and mCry2, 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).

An interlocked transcriptional-translational feedback loop (TTFL) is thought to generate the mammalian circadian clockwork in both the central pacemaker residing in the hypothalamic suprachiasmatic nuclei and in peripheral tissues. The core circadian genes, including Period1 and Period2 (Per1 and Per2), Cryptochrome1 and Cryptochrome2 (Cry1 and Cry2), Bmal1, and Clock are indispensable components of this biological clockwork. The cycling of the Per and Cry clock proteins has been thought to be necessary to keep the mammalian clock ticking. This study provides a novel cell-permeant protein approach for manipulating cryptochrome protein levels to evaluate the current transcription and translation feedback model of the circadian clockwork. Cell-permeant cryptochrome proteins appear to be functional on the basis of several criteria, including the abilities to (1) rescue circadian properties in Cry1^−/−Cry2^−/− mouse fibroblasts, (2) act as transcriptional repressors, and (3) phase shift the circadian oscillator in Rat-1 fibroblasts. By using cell-permeant cryptochrome proteins, it has been demonstrated that cycling of CRY1, CRY2, and BMAL1 is not necessary for circadian-clock function in fibroblasts. These results are not supportive of the current version of the transcription and translation feedback-loop model of the mammalian clock mechanism, in which cycling of the essential clock proteins CRY1 and CRY2 is thought to be necessary (Fan, 2007).

Is it possible that there is a core mammalian clockwork that is purely posttranslational? This possibility cannot be ignored, and it is possible that the results with CP-CRY proteins intimate that underlying posttranslational clock. An intriguing final speculation upon the discrepancy between these results and the earlier literature is that artificial intracellular expression of CRY1 has the effect of continuously flooding the cells with newly synthesized protein that is not posttranslationally modified, whereas the CP-CRYs have already incorporated the FAD/pterin cofactors and may be otherwise posttranslationally modified. Perhaps if a posttranslational oscillator is the core mammalian clockwork, the introduction of posttranslationally modified CRY (as with CP-CRYs) may perturb the core oscillator less than the new synthesis of unmodified CRY proteins. It may be in the fullness of time that this era of re-evaluation will lead to minor alterations of the understanding of the mammalian clockwork and that the transcriptional-translational feedback loop model will emerge triumphant. In contrast, this time of re-evaluation may lead to a very different comprehension of the mammalian clock mechanism (Fan, 2007).

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).

Mouse mCRY1 and zebrafish zCRY1a and zCRY3 belong to the DNA photolyase/Cryptochrome family. mCRY1 and zCRY1a repress CLOCK:BMAL1-mediated transcription, whereas zCRY3 does not. Reciprocal chimeras between zCRY1a and zCRY3 were generated to determine the zCRY1a regions responsible for nuclear translocation, interaction with the CLOCK:BMAL1 heterodimer, and repression of CLOCK:BMAL1-mediated transcription. Three regions, RD-2a-(126-196), RD-1-(197-263), and RD-2b-(264-293), were identified. Proteins in this family consist of an N-terminal alpha/beta domain and a C-terminal helical domain connected by an interdomain loop. RD-2a is within this loop, RD-1 is at the N-terminal 50 amino acids, and RD-2b at the following 31 amino acid residues of the helical domain. Either RD-2a or RD-1 is required for interaction with the CLOCK: BMAL1 heterodimer, and either RD-1 or RD-2b is required for the nuclear translocation of CRY. Both of these functions are prerequisites for the transcriptional repressor activity. The functional nuclear localizing signal in the RD-2b region also was identified. The sequence is well conserved among repressor-type CRYs, including mCRY1. Mutations in the nuclear localizing signal of mCRY1 reduce the extent of its nuclear localization. These findings show that both nuclear localization and interaction with the CLOCK:BMAL heterodimer are essential for transcriptional repression by CRY (Hirayama, 2003).

Circadian rhythms are driven by molecular clocks composed of interlocking transcription/translation feedback loops. Cryptochrome proteins are critical components of these clocks and repress the activity of the transcription factor heterodimer CLOCK/BMAL1. Unlike the homologous DNA repair enzyme 6-4 PHOTOLYASE, Cryptochromes have extended carboxyl-terminal tails and cannot repair DNA damage. Unlike mammals, Xenopus laevis contains both Cryptochromes (xCRYs) and 6-4 PHOTOLYASE (xPHOTOLYASE), providing an excellent comparative tool to study Cry repressive function. Findings can be extended to CRYs in general because xCRYs share high sequence homology with mammalian CRYs. Deletion of xCRYs' C-terminal domain produces proteins that are, like xPHOTOLYASE, unable to suppress CLOCK/BMAL1 activation. However, these truncations also cause the proteins to be cytoplasmically localized. A heterologous nuclear localization signal (NLS) restores the truncation mutants' nuclear localization and repressive activity. These results demonstrate that the CRYs' C termini are essential for nuclear localization but not necessary for the suppression of CLOCK/BMAL1 activation; this finding indicates that these two functions reside in separable domains. Furthermore, the functional differences between CRYs and PHOTOLYASE can be attributed to the few amino acid changes in the conserved portions of these proteins (Zhu, 2003).

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).

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).

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).

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).

The circadian clock is driven by cell-autonomous transcription/translation feedback loops. The BMAL1 transcription factor is an indispensable component of the positive arm of this molecular oscillator in mammals. A molecular genetic screening assay for mutant circadian clock proteins is presented that is based on real-time circadian rhythm monitoring in cultured fibroblasts. By using this assay, a domain was identified in the extreme C terminus of BMAL1 that plays an essential role in the rhythmic control of E-box-mediated circadian transcription. Remarkably, the last 43 aa of BMAL1 are required for transcriptional activation, as well as for association with the circadian transcriptional repressor Cryptochrome 1 (CRY1), depending on the coexistence of Clock protein. C-terminally truncated BMAL1 mutant proteins still associate with mPER2 (another protein of the negative feedback loop), suggesting that an additional repression mechanism may converge on the N terminus. Taken together, these results suggest that the C-terminal region of BMAL1 is involved in determining the balance between circadian transcriptional activation and suppression (Kiyohara, 2006).

Direct evidence for the requirement of transcriptional feedback repression in circadian clock function has been elusive. A molecular genetic screen was developed in mammalian cells to identify mutants of the circadian transcriptional activators Clock and BMAL1, which were uncoupled from Cryptochrome-mediated transcriptional repression. Notably, mutations in the PER-ARNT-SIM domain of Clock and the C terminus of BMAL1 result in synergistic insensitivity through reduced physical interactions with Cry. Coexpression of these mutant proteins in cultured fibroblasts causes arrhythmic phenotypes in population and single-cell assays. These data demonstrate that Cry-mediated repression of the Clock/BMAL1 complex activity is required for maintenance of circadian rhythmicity and provide formal proof that transcriptional feedback is required for mammalian clock function (Sato, 2006).

The Timeless protein is essential for circadian rhythm in Drosophila. The Timeless orthologue in mice is essential for viability and appears to be required for the maintenance of a robust circadian rhythm as well. The human Timeless protein interacts with both the circadian clock protein cryptochrome 2 and with the cell cycle checkpoint proteins Chk1 and the ATR-ATRIP complex and plays an important role in the DNA damage checkpoint response. Down-regulation of Timeless in human cells seriously compromises replication and intra-S checkpoints, indicating an intimate connection between the circadian cycle and the DNA damage checkpoints that is in part mediated by the Timeless protein (Unsal-Kacmaz, 2005).

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).

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 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 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, 2003).

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, 2003).

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, 2003).