gd9507.qxd 11/10/1999 12:14 PM Page 588 588 The ins and outs of circadian timekeeping Steven A Brown* and Ueli Schibler† Recent research in Drosophila and in mammals has generated The mechanism by which light signals entrain the clock is fascinating new models for how circadian clocks in these another topic of intense interest, and will be our other focus. organisms are reset by light and how these clocks, in turn, direct circadian outputs. Though light perception by the central clock is Central clock mechanisms: a brief summary ocular in mammals, it probably proceeds via a mechanism In all cases examined to date, circadian clocks have been separate from traditional visual transduction. In Drosophila, one cell-autonomous: a single cell can generate and maintain mechanism is non-ocular and is in fact present in many different self-sustained circadian oscillations. The molecular basis tissues. In both organisms, the cryptochrome family of for these rhythms may rely on a negative feedback loop in photoreceptor-like molecules plays a role in the circadian clock, which clock proteins negatively regulate their own abun- though their function is incompletely understood. Moreover, dance or activity. This regulation may occur both at the although a master clock resides in the brain, a functional clock transcriptional and at the post-transcriptional level. For appears to reside in most cells of the body. In these tissues, at example, in the bread mold Neurospora crassa, the Fre- least some output genes are controlled at the transcriptional quency protein negatively regulates its own transcription level directly by clock proteins; others appear to be regulated by by interfering with the ability of the White-collar-1 and cascades of circadian transcription factors. Taken together, White-collar-2 (WC-1 and WC-2) proteins to activate fre- these observations are reshaping thinking about inputs and quency transcription [3]. Similarly, the clock from the outputs of metazoan circadian clocks. cyanobacteria Synechococcus consists of a single cluster of three genes — kaiA, B, and C — whose mRNA levels Addresses oscillate in a circadian fashion and whose protein products Département de Biologie Moleculaire, Université de Genéve, Sciences govern expression of the kai locus [4•]. II, 30 quai Ernest-Ansermet, 1211 Genéve 4, Switzerland *e-mail: [email protected] Similar negative feedback loops appear to operate in meta- †e-mail: [email protected] zoans. From Drosophila melanogaster to mammals, striking Current Opinion in Genetics & Development 1999, 9:588–594 similarities in clock mechanism have been observed. In 0959-437X/99/$ — see front matter © 1999 Elsevier Science Ltd. Drosophila, the clock genes period and timeless are tran- All rights reserved. scribed in circadian fashion. This rhythm is affected by negative feedback from the Period and Timeless proteins, Abbreviations cry cryptochrome as mutations in period and timeless affect the timing of their SCN suprachiasmatic nucleus transcription. Indeed, the transcriptional rhythm is thought WC White-collar to be generated by Period and Timeless themselves, which heterodimerize and translocate to the nucleus in order to Introduction repress their own transcription [5•]. They probably do this Circadian clocks have been conserved in many instances by interfering with the ability of a protein heterodimer throughout the evolution of living organisms, allowing bac- composed of the Clock and Cycle proteins to stimulate teria, animals, and plants to adapt their physiological needs transcription via an E-box motif [6,7••]. Such E-box to the time of day in an anticipatory fashion. These pace- sequences are found in both the period and timeless promot- makers regulate a plethora of processes. In cyanobacteria, er regions. Circadian post-transcriptional regulation is also for example, the majority of promoters of metabolic genes believed to play a crucial role in clock function. For exam- appear to exhibit cyclical expression [1]. In mammals, ple, the protein Double-time phosphorylates Period physiological processes like sleep–wake cycles, body tem- protein, which in turn probably enhances the proteolytic perature, heartbeat, and many aspects of liver, kidney, and degradation of Period [8•,9•]. As shown in Figure 1, virtual- digestive tract physiology are all under circadian control ly all of the Drosophila proteins and sequence motifs [2]. The fashion in which clocks control such diverse func- discussed above possess one or more mammalian homologs tions in metazoan organisms remains a largely unexplored that have been suggested to play similar roles in mam- question in the field of chronobiology, and will be one malian function. A possible exception is the recently focus of our review. discovered mammalian homolog of timeless, whose mRNA expression — low-level and constant — is quite different Circadian clocks continue to ‘tell time’ in the absence of from the cyclically expressed Drosophila timeless gene, and environmental time cues such as changes in light intensity. whose in vivo functions remain unclear [10•,11•]. The period length (‘Tau’ or ‘τ’ in clock parlance) of each cycle is approximately, 24 hours under constant conditions Communication between the central (e.g. constant darkness). Hence, in order for the circadian pacemaker and peripheral clocks clock to tell time accurately, it must be readjusted (or phase Complex multicellular organisms appear to have central shifted) every day by the light regimen (or photoperiod). clocks that reside in discrete ‘pacemaker tissues’ in the gd9507.qxd 11/10/1999 12:14 PM Page 589 The ins and outs of circadian timekeeping Brown and Schibler 589 central nervous system. In these tissues, individual inde- Figure 1 pendent clocks in each cell cooperate to generate an as-yet-unknown timing signal used by the body as a whole. In Drosophila, these are composed of eight lateral (a) Drosophila neurons in the brain [12]. In lower vertebrates, the pineal gland can play a similar role, and in mammals the suprachiasmatic nucleus (SCN) of the hypothalamus is Clock Cycle the primary pacemaker tissue. Physiological studies led to the simple model that signals from the pacemaker tis- E-box period, timeless sues direct circadian output responses in peripheral tissues areas [13]. Recent evidence, however, suggests that the actual mech- P Double-time P Period anism may be more complicated. In flies, fish, and ? P Timeless mammals, circadian clocks independent of canonical pacemaker tissues have been found to be much more widespread. Circadian rhythms of melatonin synthesis (b) Mammals were found in cultured retinas of many vertebrate species [14,15] and circadian rhythms of Period protein synthesis were discovered in cultured Drosophila pupal ring glands [16]. Independent clocks were then found in explanted Clock Bmal1 tissues throughout the Drosophila body, including head, E-box period 1, period 2 thorax, and abdominal structures [17], and in isolated and period 3 zebrafish organs such as kidney and heart [18••]. Finally, circadian rhythms of clock gene expression were found to be induced by a serum shock administered to serum- P •• Casein kinase Period starved immortalized rat fibroblasts in culture [19 ]. ε P 1 P Thus, current evidence suggests that most cells may have ? Heterodimers Period–Period and possibly a circadian clock. Period–Timeless At first glance, the utility of separate clocks in individual Current Opinion in Genetics & Development peripheral cells is difficult to explain. It is possible, how- Clock molecules and models of clock mechanisms in (a) Drosophila ever, that this redundancy has evolved for more efficient and in (b) mammals. In both cases, Period proteins heterodimerize (in control of circadian outputs. Different outputs are activat- Drosophila, with Timeless protein; in mammals, the relevant partners ed at different times. In order to control all of them remain to be established) to repress their own transcription. This individually, the SCN (or other central pacemaker tissues) transcription is activated by heterodimers of Clock and Cycle (in would have to emit separate controlling signals for each Drosophila) or Clock and Bmal1 (in mammals), which act through E-box motifs in the promoter region. Accumulation of Period protein in differently phased output. By contrast, if each cell has its Drosophila is attenuated by phosphorylations effected by the Double- own clock, the pacemaker needs to send only one syn- time protein, a homolog of the mammalian casein kinase 1ε. chronizing signal periodically and peripheral clocks might then be able to function autonomously to regulate circadi- an outputs. Such a synchronizing signal is probably of such a strategy. The product of this gene is responsible humoral rather than neuronal as circadian rhythms in for the circadian regulation of salt and water balance in the expression of the Period 2 gene have been detected in bloodstream. The vasopressin promoter region harbors an cells that are not connected to the peripheral nervous sys- E-box motif similar to the one thought to mediate tran- tem, such as leucocytes [20]. Figure 2 illustrates two scriptional activation of the period genes via heterodimers of alternative models by which central pacemakers could the Clock and Bmal proteins, and vasopressin transcription direct overt biochemical outputs in the periphery. Obvi- is drastically reduced in homozygous clock mutant mice ously, these two models need not be mutually exclusive. [21].