Molecular Bases for Circadian Clocks

Life is a cyclical chemical process that is regulated in four dimensions. We distinguish parts of the cycle: development describes the changes from single cell to adult, and aging the changes from adult to death. Birth to death, a cycle, and there are cycles within cycles—circannual rhythms, menstrual cycles, semilunar cycles, and daily 24 hr or circadian cycles. Twice a year we get a reminder of the importance of our internal circadian biological clocks. Daylight savings: in October we fall back just an hour, and yet we wake up an hour early on Monday anyway and think meals are late—but only for a day, until our clocks are reset. The reminder is about the way we process environmental information and time, namely that we use external time cues (light and temperature changes that track the day without) to set an internal clock that guides the day within. This internal clock is the lens through which we survey acute external factors; it takes the lead in determining what we perceive as time. It used to be that research in chronobiology moved along at a gentlemanly pace. It was a field in that it shared a common set of problems, a common vocabulary, and a series of common assumptions: only eukaryotes had real clocks and they probably evolved just once, since the basic properties of the rhythms were generally the same. Any cell in fungi, plants, or protists could be a clock cell, but only neurons kept time in organisms that had them. Input to the clock was readily separable from how the clock itself worked. But within the past few years progress in understanding how clocks work in this assemblage of organisms has been increasing exponentially, coming to a crescendo during the final half of 1998 in an eruption of data that has largely disproven the assumptions and permanently changed the face of the field. The dust is still settling, but what we now see, albeit in broad outline, is probably the outline of how a large part of biological timing works at the molecular level. It’s been quite a ride. How’d we get this far? One ought naturally to be able to consult reviews, but there have been so many concerning the molecular analysis of rhythms that clearly what is needed here is a review of the survey of the reviews. This brings to mind a short story by H. L. Mencken in which peace of mind was brought to the literary populace in the early 1900s only through the synthesis and condensation of all of the pertinent literary critiques each week into reviews of reviews and ultimately into a grand review word (the first week being something like MIFLHMP) that readers could read, be satisfied that they were up to date, and enjoy their evenings being at home, content (90Mencken, H.L. (1919). The criticism of criticism of criticism. In Prejudices—First Series, H.L. Mencken, ed. (New York: Knopf), pp. 308–316.Google Scholar). For such a telegraphically quick review of the molecular basis of the currently understood transcription/translation feedback loop (we’ll get to this) circadian oscillators, the review word for the late 80s and early 90s would have been PERFRQT, reflecting the Drosophila period gene and the Neurospora frequency gene (the fruits of the first decades of genetic and molecular genetic analysis of clocks) and the fact that the Drosophila timeless gene, tim, was still in the process of arriving. This era was spent convincing ourselves that such genes really were the key to understanding how clocks work. Flies and fungi were PERFRQT systems for working out basic tools, paradigms, and approaches—gene products whose expression levels themselves oscillate, the importance of negative feedback, criteria to begin to distinguish which oscillatory gene products might contribute to the action of an internal timer as distinct from being output (reviewed in 32Dunlap J.C Genetic and molecular analysis of circadian rhythms.Annu. Rev. Genetics. 1996; 30: 579-601Crossref PubMed Scopus (167) Google Scholar), and a universal appreciation of the importance of genetics. If overall this left us with a less than PERFRQT understanding of timing in general, at least many found optimism in the sense that we were, finally, asking the right questions. This naturally segued into an interlude where light resetting was explained by two different mechanisms, through transcriptional induction of oscillator components in Neurospora (26Crosthwaite S.C Loros J.J Dunlap J.C Light-Induced resetting of a circadian clock is mediated by a rapid increase in frequency transcript.Cell. 1995; 81: 1003-1012Abstract Full Text PDF PubMed Scopus (242) Google Scholar) or protein turnover in Drosophila (reviewed in 166Young M The molecular control of circadian behavioral rhythms and their entrainment in Drosophila.Annu. Rev. Biochem. 1998; 67: 135-152Crossref PubMed Scopus (137) Google Scholar). But by mid 1997 the word was PASWCCLK (the first clock components with known biochemical functions [transcriptional activators], the first mammalian clock gene, and the first protein domain [PAS] conserved among clock molecules from different phyla) and then MPERMPER (mammalian orthologs and paralogs of model system clock genes), and then in mid 1998 the already ungainly CYCBMALJRKDBT (and a grand unifying theory for clocks within the animal/fungal clade of the crown eukaryotes; e.g., 34Dunlap J.C An end in the beginning.Science. 1998; 280 (b): 1548-1549Crossref PubMed Scopus (76) Google Scholar), and for the close of 1998 [WHAT WORD?]. So if you can be satisfied by intoning the four words in a dimly lit room, then enjoy your evening; and if not, read on to find out who’s who in the phylogeny of timers. It is now common to begin from a general assertion that, at the most basic level, circadian oscillators (but not systems) will be describable as a circular list of causes and effects that closes within the bounds of a single cell, even in the most complicated systems like the vertebrate suprachiasmatic nucleus (SCN) (reviewed in 16Block G.D Geusz M Khalsa S Michel S A clockwork Bullacellular study of a model circadian system.Semin. Neurosci. 1995; 7: 37-42Crossref Google Scholar, 160Welsh D.K Logothetis D.E Meister M Reppert S.M Individual neurons dissociated from rat suprachiasmatic nucleus express independently phased circadian firing rhythms.Neuron. 1995; 14: 697-706Abstract Full Text PDF PubMed Google Scholar, 58Herzog E Takahashi J Block G Clock controls circadian period in isolated suprachiasmatic nucleus neurons.Nature Neurosci. 1998; 1: 708-713Crossref PubMed Google Scholar). Events that happen outside the cell, or interactions of the cell with surrounding cells (e.g., 80Liu C Weaver D Strogatz S Reppert S Cellular construction of a circadian clock period determination in the suprachiasmatic nuclei.Cell. 1997; 91 (a): 855-860Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar) or the environment will influence the behavior of the clock-in-cell, but they are not necessary to describe its progress. However, events outside clock cells will affect the clock’s progress, thus giving rise to the distinction between the circadian oscillator and the circadian system. Here, the oscillator is taken to mean the minimal set of molecular causes and effects sufficient to describe circadian cycles as they might operate (i.e., what you’d want to add in a reconstruction experiment to make it go). There are three general questions which, if answered in terms of genetics and biochemistry, would adequately describe this clock. The first question revolves around “How does the clock work,” meaning what is the biochemical and genetic basis for the oscillator that lies at the base of the observed rhythms. A second set of questions concerns input—how this intracellular oscillator is brought into synchrony with the geophysical cycles of the extracellular, and extraorganismal, world. Third, given a synchronizable intracellular clock, how is the “molecular time” generated by the clock then transduced within the cell to bring about changes in the behavior of the cell, and thereby bring about changes in the behavior of the organism; this is output. It is clear, especially in vertebrates, that there is feedback from the output behavior back to the clock (e.g., 100Mrosovsky N Reebs S.G Honrado G.I Salmon P.A Behavioral entrainment of circadian rhythms.Experientia. 1989; 45: 696-702Crossref PubMed Google Scholar), from the clock to input photoreceptors (e.g., 40Fleissner G Fleissner G Feedback loops in the circadian system.Disc. Neurosci. 1992; 8: 79-84Google Scholar), and from output to input ion channels surrounding and affecting the clock but not being necessary for its basic timing (e.g., 16Block G.D Geusz M Khalsa S Michel S A clockwork Bullacellular study of a model circadian system.Semin. Neurosci. 1995; 7: 37-42Crossref Google Scholar). The ensemble of these interactions will be needed to perfectly model the circadian systems of real life; however, this narration would circumscribe more than what is my goal here, which is simply to cartoon the core oscillator(s) in clock cells. Some 30 years ago, Colin Pittendrigh penned for a Harvey lecture that “a truly general biology is an evolutionary biology” (112Pittendrigh C.S On temporal organization in living systems.The Harvey Lectures. 1961; 56: 93-125Google Scholar). By this he meant that evolution provides a great perspective for viewing any biological problem, one that emphasizes that the organization one sees in a system now is strongly dependent both upon physical necessities (which lead to convergent evolution) and upon previous choices made during evolution that delimit later options. That circadian clocks are adaptive is apparent (and recently proven [110Ouyang Y Andersson C.R Kondo T Golden S.S Johnson C.H Resonating circadian clocks enhance fitness in cyanobacteria.Proc. Natl. Acad. Sci. USA. 1998; 95: 8660-8664Crossref PubMed Scopus (296) Google Scholar]), but one of the aspects of chronobiology that so fascinates chronobiologists is the extent to which the problem itself, really one of basic self-controlling intracellular regulation, keeps luring students of time back out of the lab to consider the real world of light/dark cycles, of cyclic food availability, and of predation. We remain a long way from a true evolutionary biology of biological timing, but Figure 1 may provide a frame of reference through which to discern some trends. First, clocks have been sought in all three kingdoms, albeit only sporadically within the Archaebacteria (where none have been found). They exist in some cyanobacteria (reviewed in 48Golden S Johnson C.H Kondo T The cyanobacterial circadian system A clock apart.Curr. Opin. Microbiol. 1998; 1: 693-697Crossref PubMed Google Scholar) but apparently not in most Eubacteria, are found frequently among the Eukaryota, and are nearly ubiquitous among the taxa that emerged during the Cambrian phylogenetic explosion and that comprise the “Crown Eukaryotes” of the lineage, the Plantae, Fungi, and Animalia (138Sogin, M.L. (1994). The origin of eukaryotes and evolution into major kingdoms. In Early Life on Earth. Nobel Symposium No. 84, S. Bengston, ed. (New York: Columbia Univ. Press), pp. 181–192.Google Scholar). At first glimpse, the sightings of rhythmicity on this tree suggest the possibility of more than one but not dozens of independent origins for timing. However, support for such a conjecture in sequence data may be hard to come by because, perhaps reflecting their close interface with the environment, clock genes are among the most rapidly evolving genes in the organism. Be that as it may, I’ll use the Tree of Life to provide some perspective on how much and how little we know, and to focus the discussion about how living things keep time. If trends exist in the logic and molecules underlying the assembly of biological timing systems across all phyla, these trends ought to reflect the evolutionary histories of the organisms. In the accepted pattern of evolution within the terminal branches of the Tree of Life, animals and fungi share a common lineage that separated from the plants perhaps 700 million years ago, the fungi and animals subsequently diverged within a hundred million years after that, and insects from the lineage that gave rise to mammals millions of years after that. Applying the available molecular data on clocks to this phylogenetic framework, we can see common elements that may be conserved in the logic of the oscillators, in the sequences of molecules used in the oscillators, as well as in their functions within oscillatory loops—elements that are common to all living clocks, common just to the fungi and animals, common only to animals, and unique to mammals. Figure 2 is meant to provide a view of what some of the common elements might be in the logic underlying the assembly of circadian oscillators, and Table 1 a list of who’s who at the molecular level.Table 1Circadian Clock Genes: Roles, Products, and RegulationSystemGeneClock RoleProtein Product(s)RegulationPhenotype of MutantsSynechococcusaA cyanobacterial system displaying a transcription/translation–based negative feedback oscillator.kai Apositive elementno structural motifs identifiedCR—RNA peaks ∼CT 9–12; no protein datalong period (30 hr, 33 hr), ARRkai Bunknownno structural motifs identifiedCR—RNA peaks ∼CT 9–12; no protein datashort period (21 hr, 22 hr)kai Cnegative elementATP and GTP binding sitesCR—RNA peaks ∼CT 9–12; no protein data14 alleles; long, short, and ARRNeurosporabA fungal system displaying a negative feedback transcription/translation–based oscillator and using heterodimeric PAS domain–containing transcription factors as positive elements.frqnegative elementtwo proteins made from single open reading frame via temperature-responsive translational control; rhythmically phosphorylatedCR—RNA peaks ∼CT 4, and is induced by light; protein peaks ∼CT 8–12;long period (24 hr, 28 hr) alleles show loss of temperature compensation; short period alleles (16 hr, 19 hr); ARR alleleswc-1positive element; required to activate frq transcriptiontranscription factor: Zn finger DNA binding domain, GLN-rich activation domain, PAS domains mediate heterodimerization with WC-2transcriptionally induced by light; relatively constant expression in the darknull mutants and DNA binding mutants are photoblind; null mutants ARR, with low frq expressionwc-2positive element; required to activate frq transcriptiontranscription factor: Zn finger DNA binding domain, acidic activation domain, PAS domain mediates protein–protein interactions with WC-1not induced by light; protein always present in the darknull mutants photoblind and ARR, low frq expression; partial loss of function yields long period length, altered temperature compensationDrosophilacAn insect system displaying a negative feedback transcription/translation–based oscillator, using heterodimeric PAS domain–containing proteins as positive elements, and having paired negative elements.pernegative elementPAS domains mediate interaction with negative element TIM; rhythmically phosphorylatedCR—RNA peaks ∼CT 14; protein peaks ∼CT 19several long period alleles showing loss of temperature compensation; short period alleles; several ARRtimnegative elementno PAS domains; interacts with PER; phosphorylatedCR—RNA peaks ∼CT 14; protein peaks ∼CT 19long period length, short period, and ARR allelesdbtfacilitating elementsequence homolog of casein kinase 1ε; required for development; regulates accumulation of PERconstitutivelong period length, short period, and ARR allelesClk (Jrk)positive elementtranscription factor: bHLH DNA-binding domain, GLN-rich activation domain; PAS domains mediate heterodimerization with CYC; molecular relative of mammalian CLOCKCR in RNA is sometimes unimodal peaking at ca. CT23 or bimodal with a second peak near dusknull mutants ARR, show low per and tim expression; no light-induced “startle” responsecycpositive elementtranscription factor; bHLH DNA-binding domain, GLN-rich activation domain; PAS domains mediate heterodimerization with CYC; molecular relative of mammalian BMAL1/MOP3constitutivenull mutants ARR, show low per and tim expression; no light-induced “startle” responseMousedA mammalian system displaying a negative feedback transcription/translation–based oscillator, using heterodimeric PAS domain–containing proteins as positive elements, and a gene family of three similar negative elements.Per1(putative) negative elementcontains PAS domains which mediate interaction with other mammalian PER proteins; significance of interactions with negative element TIM controversial; molecular relative of insect perclear CR in mRNA expression with peak around CT4 in SCN, around CT10 in retina and peripheral tissues; transiently induced by lightno mutants availablePer2(putative) negative elementcontains PAS domains which mediate interaction with other mammalian Per proteins; significance of interactions with negative element TIM controversial; molecular relative of insect perclear CR in mRNA expression with peak around CT8 in SCN, around CT14 in retina and peripheral tissues; transiently induced by lightno mutants availablePer3(putative) negative elementcontains PAS domains which mediate interaction with other mammalian Per proteins; significance of interactions with negative element TIM controversial; molecular relative of insect perclear CR in mRNA expression with peak around CT6 in SCN, a broad peak ∼CT10–14 in retina and peripheral tissuesno mutants availabletimfacilitating (negative?) elementclear sequence homolog of insect TIMnot rhythmically or weakly rhythmically expressedno mutants availableClockpositive elementtranscription factor: bHLH DNA-binding domain, GLN-rich activation domain; PAS domains mediate heterodimerization with BMAL1/MOP3; molecular relative of insect dCLKnot rhythmically expressedone allele with long period length; homozygote has very long period, grading to ARR; reduced light induction of Per1bmal1/mop3positive elementtranscription factor: bHLH DNA-binding domain, GLN-rich activation domain; PAS domains mediate heterodimerization with CLOCK; molecular relative of insect CYCrhythmically expressed in rats; may not be rhythmically expressed in miceno mutants availableContents of this table were restricted to those genes and proteins with known or expected roles in circadian oscillatory loops.CT, circadian time; a formalism for comparing subjective time from organisms having different endogenous periodicities. By convention, CT 0, subjective dawn, and CT 12, subjective dusk. CR, circadian rhythm observed in level of expression. ARR, arrhythmic.a A cyanobacterial system displaying a transcription/translation–based negative feedback oscillator.b A fungal system displaying a negative feedback transcription/translation–based oscillator and using heterodimeric PAS domain–containing transcription factors as positive elements.c An insect system displaying a negative feedback transcription/translation–based oscillator, using heterodimeric PAS domain–containing proteins as positive elements, and having paired negative elements.d A mammalian system displaying a negative feedback transcription/translation–based oscillator, using heterodimeric PAS domain–containing proteins as positive elements, and a gene family of three similar negative elements. Open table in a new tab Contents of this table were restricted to those genes and proteins with known or expected roles in circadian oscillatory loops. CT, circadian time; a formalism for comparing subjective time from organisms having different endogenous periodicities. By convention, CT 0, subjective dawn, and CT 12, subjective dusk. CR, circadian rhythm observed in level of expression. ARR, arrhythmic. The nature of an oscillation is that it describes a system that tends, in a regular manner, to move away from equilibrium before returning. To achieve this, all that is needed is a process whose product feeds back to slow down the rate of the process itself (a negative element), and a delay in the execution of the feedback (Figure 2). Thus, biological oscillators could be built using a number of different regulatory schemes—a metabolic pathway or an ion flux should work as well as transcription and/or translation—or kinds of delay, which could result from a threshold phenomenon preventing immediate feedback (a relaxation oscillator such as a pipette washer) or from hysteresis (a slowness of response yielding an overshoot when approaching equilibrium) or nonlinearity (as when multiple components must find each other prior to executing feedback). A further necessity for a biological oscillator is a positive element, a source of excitation or activation that keeps the oscillator from winding down. Intriguingly, all known circadian oscillators use loops that close within cells (none require cell–cell interactions), and that rely on positive and negative elements in oscillators in which transcription of clock genes yields clock proteins (negative elements) which act in some way to block the action of positive element(s) whose role is to activate the clock gene(s). Figure 2 shows such an oscillator schematically and includes the names of some of the cognate elements identified in different circadian systems currently under study. This picture could be taken as implying that circadian oscillators will be simple transcription/translation feedback loops, but they will not; this is just what is in common about what has been described so far in the feedback loops that are generally (but not universally [71Lakin-Thomas P Choline depletion, frq mutations, and temperature compensation of the circadian rhythm in Neurospora crassa.J. Biol. 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In the cyanobacterium Synechococcus this is through the action of the kai A gene product, and in Crown Eukaryotes (with examples here from Neurospora, Drosophila, and mouse) it is apparently through the binding of transcriptional activators, paired by virtue of interactions via PAS domains, on the clock gene promoters. Functionally similar PAS domain–containing DNA-binding clock elements (or putative clock elements) have been described in the three best molecularly studied eukaryotic clock systems, Neurospora (27Crosthwaite S.C Dunlap J.C Loros J.J Neurospora wc-1 and wc-2transcription, photoresponses, and the origins of circadian rhythmicity.Science. 1997; 276: 763-769Crossref PubMed Scopus (341) Google Scholar), Drosophila (3Allada R White N.E So W.V Hall J.C Rosbash M A mutant Drosophila homolog of mammalian CLOCK disrupts circadian rhythms and transcription of period and timeless.Cell. 1998; 93: 805-814Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar, 29Darlington T.K Wager-Smith K Ceriani M.F Stankis D Gekakis N Steeves T Weitz C.J Takahashi J Kay S.A Closing the circadian loop CLOCK induced transcription of its own inhibitors, per and tim.Science. 1998; 280: 1599-1603Crossref PubMed Scopus (523) Google Scholar, 122Rutila J.E Suri V Le M So W.V Rosbash M Hall J.C CYCLE is a second bHLH-PAS clock protein essential for circadian rhythmicity and transcription of Drosophila period and timeless.Cell. 1998; 93: 805-813Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar), and mouse (46Gekakis N Stankis D Nguyen H.B Davis F.C Wilsbacher L.D King D.P Takahashi J.S Weitz C.J Role of the CLOCK protein in the mammalian circadian mechanism.Science. 1998; 280: 1564-1569Crossref PubMed Scopus (931) Google Scholar, 60Hogenesch J.B Gu Y.-Z Jain S Bradfield C.A The basic-helix-loop-helix-PAS orphan MOP3 forms transcriptionally active complexes with circadian and hypoxia factors.Proc. 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Since as the loop cycles it generates cyclical inhibition of transcription factors (the positive elements), the action of these positive elements on other clock-controlled genes provides an appealing idea for the escapement by which time information from the oscillator might drive output by virtue of regulating target clock-controlled genes (ccg s) (61Honma S Ikeda M Abe H Tanahashi Y Narmihira M Honma K Normura M Circadian oscillation of BMAL1, a partner of a mammalian clock gene Clock, in rat suprachiasmatic nucleus.Biochem. Biophy. Res. 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(A potential caveat here might have been that the original rhythm-mutant screens targeted nonessential genes; however, more recently screens in flies and fungi have not been biased against lethals and yet they continue to turn up new mutations in old loci. Perhaps we may be closing in on a full list.) Although not all of the details of all of the above have been described yet in all systems from cyanobacteria through fungi through humans, many of these elements are known in all of the systems examined, and the threads of similarity among all systems suggest that this emerging th