We determined that two mouse cryptochrome genes, mCry1 and mCry2, act in the negative limb of the clock feedback loop. In cell lines, mPER proteins (alone or in combination) have modest effects on their cellular location and ability to inhibit CLOCK:BMAL1 -mediated transcription. This suggested cryptochrome involvement in the negative limb of the feedback loop. 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.

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 had severely disrupted locomotor activity rhythms during extended exposure to constant darkness. Clock gene RNA rhythms were blunted in the suprachiasmatic nucleus of mPer2 mutant mice, but not of mPER1-deficient mice. Peak mPER and mCRY1 protein levels were reduced in both lines. Behavioral rhythms of mPer1/mPer3 and mPer2/mPer3 double-mutant mice resembled 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 were 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.

We examined the transcriptional regulation of the clock-controlled arginine vasopressin gene in the suprachiasmatic nuclei (SCN). 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. We show that 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. These data indicate that the transcriptional machinery of the core clockwork directly regulates a clock-controlled output rhythm.

The pineal hormone melatonin elicits two effects on the suprachiasmatic nuclei (SCN): acute neuronal inhibition and phase-shifting. Melatonin evokes its biological effects through G protein-coupled receptors. Since the Mel1a melatonin receptor may transduce the major neurobiological actions of melatonin in mammals, we examined whether it mediates both melatonin effects on SCN function by using mice with targeted disruption of the Mel1a receptor. The Mel1a receptor accounts for all detectable, high affinity melatonin binding in mouse brain. Functionally, this receptor is necessary for the acute inhibitory action of melatonin on the SCN. Melatonin-induced phase shifts, however, are only modestly altered in the receptor-deficient mice; pertussis toxin still blocks melatonin-induced phase shifts in Mel1a receptor-deficient mice. The other melatonin receptor subtype, the Mel1b receptor, is expressed in mouse SCN, implicating it in the phase-shifting response. The results provide a molecular basis for two distinct, mechanistically separable effects of melatonin on SCN physiology.

Neurons in the mammalian suprachiasmatic nucleus (SCN) contain a cell-autonomous circadian clock that is based on a transcriptional-translational feedback loop. The basic helix-loop-helix-PAS proteins CLOCK and BMAL1 are positive regulators and drive the expression of the negative regulators CRY1 and CRY2, as well as PER1, PER2, and PER3. To assess the role of mouse PER3 (mPER3) in the circadian timing system, we generated mice with a targeted disruption of the mPer3 gene. Western blot analysis confirmed the absence of mPER3-immunoreactive proteins in mice homozygous for the targeted allele. mPer1, mPer2, mCry1, and Bmal1 RNA rhythms in the SCN did not differ between mPER3-deficient and wild-type mice. Rhythmic expression of mPer1 and mPer2 RNAs in skeletal muscle also did not differ between mPER3-deficient and wild-type mice. mPer3 transcripts were rhythmically expressed in the SCN and skeletal muscle of mice homozygous for the targeted allele, but the level of expression of the mutant transcript was lower than that in wild-type controls. Locomotor activity rhythms in mPER3-deficient mice were grossly normal, but the circadian cycle length was significantly (0.5 h) shorter than that in controls. The results demonstrate that mPer3 is not necessary for circadian rhythms in mice.

The hormone, melatonin, is produced rhythmically in the vertebrate pineal gland (13). Rhythmic melatonin production plays a critical role in the regulation of reproduction in seasonally breeding mammals (1, 24). In nonmammalian vertebrates, melatonin also plays a major role in the regulation of circadian rhythms (4, 46). While endogenous melatonin appears to play only a subtle role in the regulation of circadian rhythms in mammals, exogenous melatonin influences circadian rhythms in several rodent species and in humans (2,3,9,21,22,39,46,47). While the magnitude of this phase shifting effect is small for adult mammals, it is nevertheless critical in some situations; e.g., daily melatonin administration appears to be useful for entrainment of non-24-h circadian cycles to the 24-hour day for blind individuals (22).Two high-affinity receptors for melatonin have been identified in mammals (for reviews, see references 33 and 43). These receptors, the Mel 1a and Mel 1b receptor subtypes, are members of the G protein-coupled receptor superfamily (29,30). A melatonin receptor-related receptor (also called H9 and GPR50) has sequence homology to the Mel 1a and Mel 1b receptors but does not bind melatonin (7,16,32). In addition to these three members of the gene family in mammals, nonmammalian species have a third high-affinity melatonin receptor subtype, the Mel 1c receptor (31). The mammalian Mel 1a and Mel 1b receptors are also called the MT1 and MT2 receptors, respectively [10]. The inability of this MT nomenclature system to accommodate the nonmammalian Mel 1c receptor has resulted in persistence of dual nomenclature systems; here, we use the original nomenclature.The relative importance of the mammalian melatonin receptor subtypes in mediating circadian responses to the hormone is unclear. The circadian clocks of neonatal hamsters are very efficiently set by even a single injection of melatonin, and several physiological and in vitro responses of the suprachiasmatic nucleus (SCN) to melatonin have been observed in hamsters (25,36,39,47). Remarkably, however, the Mel 1b receptor gene of several hamster species, including those with robust circadian and reproductive responses to the hormone, does not encode a functional melatonin receptor (47; see also GenBank accession number AY145849). Pharmacological studies with mice, however, suggest that the mouse Mel 1b receptor mediates circadian responses (9,19). Other data, derived from mice with targeted disruption of the Mel 1a melatonin receptor, indicate that this subtype mediates an acute suppressive effect of melatonin on SCN neuronal firing, and they reveal apparent redundancy of receptor subtypes in mediating the phase shifting response to melatonin (23).To determine whether the mouse Mel 1b receptor contributes

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We cloned the mouse cDNA of a mammalian homolog of the Drosophila timeless (tim) gene and designated it mTim. The mTim protein shows five homologous regions with Drosophila TIM. mTim is weakly expressed in the suprachiasmatic nuclei (SCN) but exhibits robust expression in the hypophyseal pars tuberalis (PT). mTim RNA levels do not oscillate in the SCN nor are they acutely altered by light exposure during subjective night. mTim RNA is expressed at low levels in several peripheral tissues, including eyes, and is heavily expressed in spleen and testis. Yeast two-hybrid assays revealed an array of interactions between the various mPER proteins but no mPER-mTIM interactions. The data suggest that PER-PER interactions have replaced the function of PER-TIM dimers in the molecular workings of the mammalian circadian clock.