using transgenic animals that used the promoters of different clock genes to drive the expression of reporters such as luciferase and green fluorescent protein.

All of these approaches have led to the observation that clock genes are expressed outside the SCN [78] and indeed outside the brain [79]. Furthermore, clock gene expression is rhythmic in many of these tissues, including the liver, testes, and skeletal muscles [79]. It is possible that oscillations in gene expression in these peripheral tissues require the SCN as animals with SCN lesions do not exhibit circadian oscillations in gene expression in their liver [80]. This could be due to the loss of a daily organizing signal from the SCN, which maintains synchrony among the individual oscillating cells in these peripheral tissues. Cultured lung, skeletal muscle, liver, and SCN cells all exhibit circadian rhythms in gene expression as determined by real-time fluorescent reporters from a luciferase transgene driven by a promoter for one of the clock genes [81]. Even cultured fibroblasts can exhibit a circadian rhythm in gene expression if they are treated with a serum shock [82]. In all of these cultures, except for those of the SCN, oscillations decrease in amplitude and eventually extinguish after a number of cycles [81, 82]. This is believed to occur because, while each individual cell in these cultures continues to oscillate, synchrony among these oscillating cells is gradually lost [82–84]. Rhythmicity in these peripheral organs likely helps coordinate the diverse biochemical tasks carried out by these organs, such that incompatible biochemical reactions occur at different times, and metabolic reactions with high energetic demands are limited to only those times of the day when they are necessary [85].

CLOCK GENES

The last decade or so has witnessed an explosion in our understanding of the molecular underpinnings of the mammalian circadian system, with the discovery of at least nine clock genes [1]. Our understanding of how these genes interact to yield the molecular circadian clock continues to evolve as recent findings have forced the field to reconsider the roles played by two of these genes [86, 87]. We now know that the circadian clock ticks at the level of single cells [31], and that this clock is a product of interlocked positive and negative transcription–translation feedback loops.

While a genetic basis for circadian rhythmicity had long been suspected due to period mutants in fruit flies and Syrian hamsters as well as strain differences in period in mice, it was not until 1994 that the first clock gene was cloned in mammals. Clock was identified using a forward genetic approach by which colonies of mice were exposed to the mutagen ethylnitrosourea (ENU) and then screened for circadian rhythm abnormalities [88]. This gene is autosomal semidominant, causing lengthening of the circadian period from 23.3h in wild-type animals, to 24.4h in heterozygous animals, and to 27.2h in homozygous animals. Homozygous Clock mutant animals quickly become arrhythmic in constant conditions. This gene codes for a basic helix-loop-helix (bHLH) transcription factor that contains a PAS dimerization domain (after the first three proteins found to contain such a domain: PER-ARNT-SIM; PER for period, ARNT for aryl hydrocarbon receptor nuclear translocator and SIM for single-minded protein). Clock dimerizes with BMAL1 (also known as MOP3) and together they act as positive regulators of genes that contain an E- box (the DNA sequence CACGTG) in their promoter [89] (Fig. 3). Both of these genes

Fig. 3. Schematic of the autoregulatory transcription–translation feedback loops that underlie the intracellular circadian clock. During the day, CLOCK/BMAL1 dimer drives the transcription of the Period, Cryptochrome, and Rev-erbα genes. The messenger RNA accumulates and is translated into proteins. PER and CRY proteins dimerize, and casein kinase 1ε phosphorylates PER proteins. In the late day to early night, the PER/CRY complex translocates to the nucleus (possibly with CK1ε), where it inhibits the activity of CLOCK and BMAL1 at E-boxes, thus terminating transcription. Transcription is reinitiated when the levels of PER and CRY proteins drop too low to continue to inhibit the activity of CLOCK and BMAL1. The auxiliary loop of rhythmic Rev-erbα production regulates the rhythmic production of BMAL1.

are highly conserved across phyla, and homologs have been found in a range of species, including Drosophila, in which BMAL1 is known as cycle. E-boxes regulate the expression of other clock genes as well as so-called clock-controlled genes such as vasopressin. It should be noted that while mutations of the clock gene impair the circadian clock, deletion of this gene has no effect on circadian rhythmicity [86]. The reasons for this are not

currently known, but it is possible that another bHLH protein may dimerize with BMAL1 only when CLOCK protein, mutant or otherwise, is completely absent.

The negative limb of the transcription–translation feedback loop is mediated by three Period and two Cryptochrome genes. All these genes have E-boxes in their promoters. The mammalian period gene Period1 (Per1) was first identified using a homology screen based on the sequence cloned for the Drosophila Period gene [90]. Two other homologs (Per2 and Per3) have since been discovered [79, 91, 92]. The peak levels of the messenger RNA (mRNA) for these genes are observed during the midday, with peak protein levels lagging behind by about 6 h. Mutations in either Per1 or Per2 alter various circadian properties. Per2 mutants have an abnormally short circadian period that degrades into arrhythmicity [93] and have attenuated phase delays to light [94]. Per1 mutants exhibit either shorter [94] or longer [95] periods and smaller phase advance [94]. When Per1 is constitutively expressed in transgenic rats, the period lengthens, and phase shifts to light are attenuated [96].

The Cryptochrome genes Cry1 and Cry2 were identified due to their homology with the blue light photoreceptor for the circadian systems of plants and flies. The peak levels of the mRNA for these genes are also observed during the midday. Disruption of these genes also results in period alterations, with longer periods observed when Cry2 is deleted and shorter periods observed when Cry1 is deleted [88, 97]. Animals lacking both Cry1 and Cry2 are completely arrhythmic when housed in constant darkness [88, 97].

The period and cryptochrome proteins are believed to dimerize and then translocate to the nucleus. Once in the nucleus, these complexes suppress the activity of CLOCK and BMAL1, thus terminating transcription of E-box-related genes. This suppression is largely attributed to the CRY proteins [98]. The leading hypothesis is that the CLOCK and BMAL1 are constitutively bound to the E-box, and that their presence leads to histone acetylation and thus chromatin remodeling that then permits RNA polymerase to bind to the promoter and initiate transcription of the gene. CRY is thought to inhibit the histone acetylation, which leads to chromatin changes that obscure the RNA polymerase promoter and prevent transcription of the gene [99]. As the Period and Cryptochrome genes are regulated by E-boxes, this negative-feedback loop has the effect of turning off the expression of these genes.

The rhythmic expression in the SCN of the clock genes Per1 and Per2 as well as the clock-controlled gene vasopressin follows a specific spatiotemporal pattern and does not occur simultaneously in every cell [43, 100]. Expression is initiated early in the day in the dorsomedial SCN, spreads ventrally over the course of the day, and then recedes back to the dorsomedial SCN by the end of the day. Such rhythmic expression appears to be largely restricted to the SCN shell [41].

The circadian system also has a secondary loop that regulates the expression of BMAL1. The expression of the BMAL1 gene is regulated by a ROR (retinoid-related orphan receptor) promoter sequence. Two groups of proteins compete for the ROR sequence to regulate BMAL1 expression, namely, the ROR and Rev-erb families of proteins [101]. Specifically, the RORα isoform activate expression, while Rev-erbα isoform inhibits expression. Rev-erbα is itself regulated by an E-box and is expressed rhythmically, in phase with the Period and Cryptochrome genes and in antiphase to BMAL1 [101].

One complete cycle of the transcription–translation feedback loop takes approximately 24 h. The rate of the these transcription–translation feedback loops that underlie the circadian clock are regulated by the casein kinase 1 family [102]. The first mammalian period mutation to be identified was the tau mutant hamster [103], a mutation subsequently mapped to the casein kinase 1ε (CK1ε) gene [102]. Hamsters that are heterozygous for this mutation have periods of approximately 22.2 h, while those that are homozygous for the mutation have periods of approximately 20.2 h [103]. CK1ε phosphorylates the period proteins, which alter their rates of translocation to the nucleus as well as their degradation rates. The tau mutation was initially thought to lead to decreased CK1ε-medi- ated phosphorylation of the PERIOD proteins, leading to premature peaks in PER1 and PER2 protein levels and in turn giving way to premature inhibition of CLOCK:BMAL1 activity [102]. This has been challenged by the observation that the tau mutation may actually increase phosphorylation of PER1 and PER2 and may lead to more rapid translocation to the nucleus or decreased degradation [87].

The expression of the Per1 and Per2 genes can also be regulated by input from the retina [79, 91, 92]. This induced expression is mediated by cAMP response element (CRE) elements in Per1 and Per2 promoters, which is regulated by the cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB). Light pulses that phase shift the circadian clock phosphorylate CREB [104]. The initial induced expression of Per1 and Per2 occurs primarily in the SCN core and most prominently in the region of CalB cells [41]. Following a delay, some Per1 and Per2 expression is observed in the SCN shell. The pattern of this expression is related to the direction of the phase shift that is produced by the same light pulse. Only phase-delaying light leads to Per2 expression in the shell, while only phase-advancing light leads to Per1 expression in the shell [42]. Per1 and Per2 expression are essential components of the photic response; when their expression is prevented by microinjecting antisense oligonucleotides for these two genes into the SCN, phase shifts to light are attenuated or blocked [105]. The rest of the clock genes, including Per3, do not change their expression following exposure to phase-shifting light pulses.

Manipulations that mimic light pulses also lead to increased expression of Period genes. The retina communicates with the SCN through glutamate release. NMDA microinjections to the SCN during the night produce phase shifts similar to those produced by light exposure at the same phases [59] and induce Per1 and Per2 expression [106]. Application of VIP to the SCN in vitro shifts the rhythm of electrical firing rates in a phase-dependent manner, such that early night application delays the phase of the peak firing rate, while application late in the night advances the peak to an earlier time. Such shifts in vitro are associated with increased Per1 and Per2 expression [107]. Microinjections of GRP to the SCN in vivo produce phase shifts in locomotor behavior comparable to those produced by light [52] and induce Per1 and Per2 expression in a discrete group of dorsolateral SCN cells [49].

While photic manipulations lead to an increased expression of Per1 and Per2, nonphotic manipulations have the opposite effect. Nonphotic manipulations, such as exercise and sleep deprivation, have their maximal effect at the same phase as peak rhythmic Per1 and Per2 expression (i.e., midday). These manipulations are associated with rapid downregulation of Per1 and Per2 [108]. In fact, decreasing Per1 expression