Mammalian circadian rhythms are regulated by the suprachiasmatic nucleus (SCN), and current dogma holds that the SCN is required for the expression of circadian rhythms in peripheral tissues. Using a PERIOD2::LUCIFERASE fusion protein as a real-time reporter of circadian dynamics in mice, we report that, contrary to previous work, peripheral tissues are capable of self-sustained circadian oscillations for >20 cycles in isolation. In addition, peripheral organs expressed tissue-specific differences in circadian period and phase. Surprisingly, lesions of the SCN in mPer2 Luciferase knockin mice did not abolish circadian rhythms in peripheral tissues, but instead caused phase desynchrony among the tissues of individual animals and from animal to animal. These results demonstrate that peripheral tissues express self-sustained, rather than damped, circadian oscillations and suggest the existence of organ-specific synchronizers of circadian rhythms at the cell and tissue level.

We used positional cloning to identify the circadian Clock gene in mice. Clock is a large transcription unit with 24 exons spanning approximately 100,000 bp of DNA from which transcript classes of 7.5 and approximately 10 kb arise. Clock encodes a novel member of the bHLH-PAS family of transcription factors. In the Clock mutant allele, an A-->T nucleotide transversion in a splice donor site causes exon skipping and deletion of 51 amino acids in the CLOCK protein. Clock is a unique gene with known circadian function and with features predicting DNA binding, protein dimerization, and activation domains. CLOCK represents the second example of a PAS domain-containing clock protein (besides Drosophila PERIOD), which suggests that this motif may define an evolutionarily conserved feature of the circadian clock mechanism.

During the past decade, the molecular mechanisms underlying the mammalian circadian clock have been defined. A core set of circadian clock genes common to most cells throughout the body code for proteins that feed back to regulate not only their own expression, but also that of clock output genes and pathways throughout the genome. The circadian system represents a complex multioscillatory temporal network in which an ensemble of coupled neurons comprising the principal circadian pacemaker in the suprachiasmatic nucleus of the hypothalamus is entrained to the daily light/dark cycle and subsequently transmits synchronizing signals to local circadian oscillators in peripheral tissues. Only recently has the importance of this system to the regulation of such fundamental biological processes as the cell cycle and metabolism become apparent. A convergence of data from microarray studies, quantitative trait locus analysis, and mutagenesis screens demonstrates the pervasiveness of circadian regulation in biological systems. The importance of maintaining the internal temporal homeostasis conferred by the circadian system is revealed by animal models in which mutations in genes coding for core components of the clock result in disease, including cancer and disturbances to the sleep/wake cycle.

The tau mutation is a semidominant autosomal allele that dramatically shortens period length of circadian rhythms in Syrian hamsters. We report the molecular identification of the tau locus using genetically directed representational difference analysis to define a region of conserved synteny in hamsters with both the mouse and human genomes. The tau locus is encoded by casein kinase I epsilon (CKIɛ), a homolog of the Drosophila circadian gene double-time. In vitro expression and functional studies of wild-type and tau mutant CKIɛ enzyme reveal that the mutant enzyme has a markedly reduced maximal velocity and autophosphorylation state. In addition, in vitro CKIɛ can interact with mammalian PERIOD proteins, and the mutant enzyme is deficient in its ability to phosphorylate PERIOD. We conclude that tau is an allele of hamster CKIɛ and propose a mechanism by which the mutation leads to the observed aberrant circadian phenotype in mutant animals.Daily rhythms in biochemical, physiological, and behavioral processes are regulated by biological clocks (1, 2). In natural conditions, the endogenous circadian rhythms that are generated by these clocks are synchronized (entrained) to the 24-hour cycles of the external world by time cues such as the daily light/dark cycle (2). While these clocks are found in organisms as divergent as cyanobacteria, plants, fruit flies, and mammals, there is an extraordinary degree of evolutionary conservation of the underlying generative molecular mechanisms (3-9). The first mammalian circadian gene, Clock, was cloned and characterized in mouse (10-12). Clock encodes a novel member of the basic helix-loop-helix (bHLH) PER-ARNT-SIM (PAS) family of transcription factors. Shortly following the cloning of Clock, three mouse Period orthologs, denoted mPer1 (13,14), and mPer3 (18, 19), as well as a Timeless (mTim) ortholog, were cloned (20)(21)(22)(23)(24). At the same time, another gene, BMAL1 (25), was found to encode the protein dimerization partner for CLOCK (26), and together the CLOCK/BMAL complex was shown to transactivate mPer1 via conserved E-box elements found in the promoters of Drosophila and mouse period genes (26-28). Finally, in Drosophila and mammals, the negative feedback effects of PER and TIM were shown to act at the level of the CLOCK/BMAL complex (20, 28) in a surprisingly direct manner (29, 30).

In a search for genes that regulate circadian rhythms in mammals, the progeny of mice treated with Nethyl-N-nitrosourea (ENU) were screened for circadian clock mutations. A semidominant mutation, Clock, that lengthens circadian period and abolishes persistence of rhythmicity was identified. Clock segregated as a single gene that mapped to the midportion of mouse chromosome † To whom correspondence should be addressed. * Present address: Department of Biological Sciences, Wichita State University, Wichita, KS 67260, USA.Published as: Science. 1994 April 29; 264(5159): 719-725. HHMI Author Manuscript HHMI Author Manuscript HHMI Author Manuscript5, a region syntenic to human chromosome 4. The power of ENU mutagenesis combined with the ability to clone murine genes by map position provides a generally applicable approach to study complex behavior in mammals.Progress has been made at the physiological and cellular levels in our understanding of circadian systems (1), yet the molecular mechanism of circadian clocks has not been fully elucidated (2). The isolation of "clock mutants" and the widespread requirement for protein synthesis in circadian clock systems imply that gene expression is an integral component of the oscillator (2). Recent molecular work with the Drosophila period (per) and Neurospora frequency (frq) genes suggests that a circadian cycle of per and frq transcription, respectively, may lie at the heart of the oscillator mechanism in these species (3). However, no information exists concerning the molecular elements of the clock system in mammals. In the absence of specific mechanistic information, genetics has been a powerful approach to uncover unknown elements. We report here the isolation of a mutation in the mouse that changes two central properties of circadian rhythms: the intrinsic period length and the persistence of rhythmicity. Taken together, our results define a gene, named Clock (for circadian locomotor out-put cycles kaput) that is essential for normal circadian behavior.Because the majority of clock mutants isolated in other organisms have been semidominant (4), we screened heterozygotes directly in the mouse. With the mutagen ENU, average forward mutation frequencies of 0.0015 per locus per gamete (1 in 700) can be achieved in the mouse (5). Male mice of the inbred strain C57BL/6J (B6) were treated with a single injection of ENU and after recovery of fertility were mated with untreated B6 females (6). First generation (G1) offspring would be heterozygous for any induced mutations but otherwise possess an isogenic B6 background (Fig. 1A). Normal B6 mice exhibit a robust circadian rhythm of wheel-running activity; we used this behavioral assay to screen for circadian mutants (Fig. 1B). Activity rhythms were monitored during exposure to a lightdark cycle (LD) to assess synchronization or entrainment behavior and in constant darkness (DD) to determine the circadian period of the locomotor activity rhythm (7). Laboratory mice typically have circadian periods of less than 24 hours, w...

The mammalian circadian system is a complex hierarchical temporal network which is organized around an ensemble of uniquely coupled cells comprising the principal circadian pacemaker in the suprachiasmatic nucleus of the hypothalamus. This central pacemaker is entrained each day by the environmental light/dark cycle and transmits synchronizing cues to cell-autonomous oscillators in tissues throughout the body. Within cells of the central pacemaker and the peripheral tissues, the underlying molecular mechanism by which oscillations in gene expression occur involves interconnected feedback loops of transcription and translation. Over the past 10 years we have learned much regarding the genetics of this system, including how it is particularly resilient when challenged by single-gene mutations, how accessory transcriptional loops enhance the robustness of oscillations, how epigenetic mechanisms contribute to the control of circadian gene expression, and how, from coupled neuronal networks, emergent clock properties arise. Here we will explore the genetics of the mammalian circadian system from cell-autonomous molecular oscillations, to interactions among central and peripheral oscillators and ultimately, to the daily rhythms of behavior observed in the animal.

The mouse Period2 (mPer2) locus is an essential negative-feedback element of the mammalian circadian-clock mechanism. Recent work has shown that mPer2 circadian gene expression persists in both central and peripheral tissues. Here, we analyze the mouse mPer2 promoter and identify a circadian enhancer (E2) with a noncanonical 5 -CACGTT-3 E-box located 20 bp upstream of the mPer2 transcription start site. The E2 enhancer accounts for most circadian transcriptional drive of the mPer2 locus by CLOCK:BMAL1, is a major site of DNaseI hypersensitivity in this region, and is constitutively bound by a transcriptional complex containing the CLOCK protein. Importantly, the E2 enhancer is sufficient to drive self-sustained circadian rhythms of luciferase activity in central and peripheral tissues from mPer2-E2::Luciferase transgenic mice with tissue-specific phase and period characteristics. Last, genetic analysis with mutations in Clock and Bmal1 shows that the E2 enhancer is a target of CLOCK and BMAL1 in vivo.Bmal1 gene ͉ circadian clock ͉ Clock gene ͉ luciferase ͉ Period2 locus T he mammalian circadian system is composed of a hierarchy of robustly rhythmic central and peripheral oscillators (1-3). An ensemble of coupled oscillators in the suprachiasmatic nucleus (SCN) of the hypothalamus is entrained by daily light input from the visual system, and neural and humoral output signals from the SCN coordinate the phase of independent circadian oscillators in peripheral tissues throughout the organism (1, 4). The clock mechanism is similar in cells of the SCN and periphery and consists of a network of transcriptional͞translational feedback loops (5-7). In the primary feedback loop, transcription is driven by the bHLH-PAS proteins CLOCK and BMAL1 (or MOP3), which heterodimerize and initiate transcription of three Period genes (in mice; mPer1, mPer2, and mPer3) and two Cryptochrome genes (mCry1 and mCry2) (8)(9)(10)(11)(12)(13)(14). The PER and CRY proteins then negatively feedback to repress transcription at their own promoters by acting on the CLOCK:BMAL1 complex (9,11,15,16). The primary feedback loop is modulated by a second feedback loop composed of the two retinoic acid-related orphan receptors (RORs), 18), which drive a circadian rhythm in Bmal1 transcription (11,19).Of the three Period genes, mPer2 plays a dominant role (14,20). However, in contrast to mPer1 (8, 9, 21), very little is known regarding the transcriptional regulation of mPer2 (10, 22,23) because of the absence of canonical 5Ј-CACGTG-3Ј E-box elements (24) within the 5Ј upstream regulatory region. Here, we report a comprehensive analysis of the circadian transcriptional drive of the mouse Per2 locus. Materials and MethodsDNA Constructs. A 3.3-kb EcoRI͞XbaI fragment was isolated from a Per2-positive bacterial artificial chromosome clone (1) and subcloned into the pGL3-Basic luciferase reporter vector. The primers used to generate truncated promoter constructs are given in the supporting information.Cell Culture. HepG2 cells were grown in DMEM supplemented w...

During the past four years, significant progress has been made in identifying the molecular components of the mammalian circadian clock system. An autoregulatory transcriptional feedback loop similar to that described in Drosophila appears to form the core circadian rhythm generating mechanism in mammals. Two basic helix-loop-helix (bHLH) PAS (PER-ARNT-SIM) transcription factors, CLOCK and BMAL1, form the positive elements of the system and drive transcription of three Period and two Cryptochrome genes. The protein products of these genes are components of a negative feedback complex that inhibits CLOCK and BMAL1 to close the circadian loop. In this review, we focus on three aspects of the circadian story in mammals: the genetics of the photic entrainment pathway; the molecular components of the circadian pacemaker in the hypothalamic suprachiasmatic nucleus; and the role of posttranslational regulation of circadian elements. A molecular description of the mammalian circadian system has revealed that circadian oscillations may be a fundamental property of many cells in the body and that a circadian hierarchy underlies the temporal organization of animals.