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EMBO reports VOL 6 | NO 10 | 2005 ©2005 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION930
review
review
The circadian cycle:daily rhythms from behaviour
to genes
First in the Cycles Review Series
Martha Merrow
1+,2
, Kamiel Spoelstra
1
& Till Roenneberg
2
1
University of Groningen,Haren, The Netherlands, and
2
University of Munich, Munich,Germany
The daily recurrence of activity and rest are so common as to seem
trivial. However, they reflect a ubiquitous temporal programme
called the circadian clock. In the absence of either anatomical
clock structures or clock genes, the timing of sleep and wakefulness
is disrupted. The complex nature of circadian behaviour is evident
in the fact that phasing of the cycle during the day varies widely for
individuals, resulting in extremes colloquially called ‘larks’ and
‘owls’. These behavioural oscillations are mirrored in the levels of
physiology and gene expression. Deciphering the underlying mech-
anisms will provide important insights into how the circadian clock
affects health and disease.
Keywords: circadian rhythm; entrainment; phase; oscillator; clock
EMBO reports (2005) 6,930–935.doi:10.1038/sj.embor.7400541
Introduction
‘Birds do it, bees do it, even educated fleas do it.’ Cole Porter was
speaking of love, but, save the refrain, he could just as well have
been referring to the circadian clock. Although he discussed only
animals, organisms of all phyla show circadian rhythms. Rhythmic
behaviour persists even in constant conditions, although with a
period slightly shorter or longer than the day (hence, circa diem),
demonstrating that the oscillation represents an endogenous
temporal programme.
In animals, daily rhythms are typically measured as activity ver-
sus rest, but hundreds of other parameters from the level of behav-
iour to gene expression are also oscillating with a circadian period.
Plants show numerous circadian rhythmic phenomena, including
leaf movement, growth rate and stomatal opening, as well as the
expression of much of the photosynthetic machinery. The fungus
Neurospora crassa makes asexual spores every 22 h in constant
darkness, and the prokaryotic genetic model organism—the
cyanobacterium Synechococcus—separates the incompatible
processes of photosynthesis and nitrogen fixation to opposite times
of the day, thereby solving the problem using time rather than
space as do other cyanobacteria. In short, daily biological rhyth-
micity is widespread in nature, with environmental cues (called
zeitgebers), such as light and temperature, synchronizing internal
time to the earth’s 24-h rotation.
Characteristics of a circadian clock
Several properties unify circadian systems in all organisms so far
studied. First is the self-sustained rhythm with its long period
(Fig 1A). The circadian system has been routinely probed by
plunging an organism into constant conditions, resulting in the
(initially) surprising finding that has become fundamental to the
field of clocks research: that all organisms have a remarkably pre-
cise, sustained, circadian rhythm. Persistent rhythms have been
observed in unicells (Lakin-Thomas & Brody, 2004;
Mergenhagen, 1980), as well as dissociated cells from the brain
(Welsh et al, 1995). Even Rat-1 fibroblasts (an old cell-culture
line) can be triggered to display circadian rhythms in gene expres-
sion (Balsalobre et al, 1998). Thus, the circadian period is generated
at the level of the cell.
Another typical feature of circadian clocks is compensation
with respect to spurious changes in the environment, some of
which can act as zeitgebers when changing on a regular (daily)
basis. Although the clock mechanism is essentially a collection of
biochemical reactions, it runs with the same period over a wide
range of temperatures (temperature compensation; Fig 1B,C).
However, temperature cycles in the 24-h range can synchronize
circadian clocks (Brown et al, 2002; Merrow et al, 1999). A flash of
lightning in the night will not reset the rhythm in many organisms,
and even a substantial amount of light, if supplied at certain times
during the cycle (that is, during the internal, subjective day when
light is ‘expected’) might not affect the free-running rhythm in
constant conditions (Fig 1D compared with Fig 1E).
The period of the free-running rhythm and its temperature com-
pensation have been determined in constant conditions but, in
nature, the clock is predominantly exposed to cycles of light, tem-
perature, food availability, predator presence, and so on. Many of
1
Biologisch Centrum, University of Groningen,PO Box 14,9750AA Haren,
The Netherlands
2
Institute for Medical Psychology, University of Munich, Goethestrasse 31,
80336 Munich,Germany
+
Corresponding author. Tel: +31 50 363 2064; Fax: +31 50 363 2148;
E-mail:m.merrow@rug.nl
Submitted 20 July 2005; accepted 23 August 2005
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M.Merrow et al
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these can act as zeitgebers for circadian systems, synchronizing
them through an active mechanism called entrainment (Fig 1F,G).
All zeitgebers mirror the cycle length of the earth’s rotation and
they therefore shaped circadian systems as they evolved. To probe
the mechanisms of entrainment (that is, how the internal, biologi-
cal oscillator interacts with the external, physical one), circadian
researchers often use non-24-h cycles (T cycles). As the period of
the cycle is shortened, the phase of entrainment occurs later
(Fig 1G). In longer cycles, the phase of entrainment advances. This
was first shown using lizards in temperature cycles (Hoffmann,
1963). If the cycle is too short or too long, the endogenous circadi-
an rhythm runs ‘through’, systematically reacting to the cycle
(Holst, 1939) but without getting stably caught by it. The ‘range of
entrainment’ within which the clock entrains is both species- and
zeitgeber-specific. When the period of an imposed cycle is about
one-half (one-third, one-quarter, etc.) of the endogenous period,
circadian rhythms typically ‘frequency demultiply’, occurring
once per two (three, four, etc.) external cycles (Fig 1H). All of these
entrainment properties show that circadian clocks behave as
robust oscillators rather than as hourglass timers, which are pas-
sively reset. In the latter case, with instant resetting properties, we
would not be troubled by jet lag!
Although circadian systems are all composed of cellular
clocks, there are large differences in their assembly. For example,
in plants, circadian rhythms in neighbouring cells are apparently
running at independent phases (Thain et al, 2000), whereas in
animals, they form a highly interdependent, hierarchical network
(Fig 2). A dedicated ‘pacemaker’ resides in the nervous system
that receives the environmental and temporal information from
zeitgebers (for example, dawn and dusk), processes them and
transmits this information through endogenous signals to the cel-
lular clocks in the periphery, supporting coordinated timing (for
example, liver, heart or kidney; Akhtar et al, 2001; Panda et al,
2002; Storch et al, 2002; Yoo et al, 2004). The mammalian pace-
maker is located in the suprachiasmatic nucleus (SCN) of the
hypothalamus. Thus, cellular clocks throughout the body are syn-
chronized with respect to the zeitgeber and each other, although
not necessarily with the same phase; molecular oscillations in
the SCN of mice held in constant darkness, for example, are
ahead of those in the liver by approximately 4–6 h (Balsalobre
et al, 2000). Furthermore, molecular oscillations of clock genes
in the liver can be uncoupled from the SCN by scheduled feeding
(Damiola et al, 2000).
What is it good for?
Circadian clocks allow organisms to anticipate and ‘prepare for’
environmental changes, thereby increasing their fitness. For
example, a photosynthetic machinery that is already geared up
when the first sunrays appear will harvest more energy. A conse-
quence of this logic is that the clock’s benefits are most obvious
under entrained conditions. By mixing wild-type cyanobacteria
and mutant strains with either long or short free-running periods,
systematic studies have shown that strains with an endogenous
period close to that of the entraining cycle are most successful,
whereas the strains did not out-compete each other in constant
conditions (Yan et al, 1998). Ablation of the SCN pacemaker in
mammals results in the loss of consolidated activity and rest.
Accordingly, SCN-lesioned chipmunks are more prone to predators
in the wild (DeCoursey & Krulas, 1998).
The seasonal changes of photoperiod become greater with
increasing distance from the equator, and many examples of
reproduction and physiology show photoperiodic regulation
(Elliott & Goldman, 1981). Flowering and seed production in
A
B
C
D
E
F
G
H
24 h
20 h
12 h
Fig 1 | Cartoon of a circadian rhythm in free-running and entrained
conditions.The width of the panels is 24 h, except for (G), which represents
20 h. Each bar exemplifies a bout of activity (in this case, the ‘activity’ of
spore formation in Neurospora crassa).(A) In constant darkness and
temperature (25 °C),spores develop once per 22 h.The period changes only
slightly with ambient temperature,lengthening when it is colder (20 °C; B),
and shortening when warmer (30 °C; C). The phase of the rhythm is not
changed significantly when a light pulse (flash) is delivered in the subjective
mid-day (D),whereas large phase shifts are induced when light is given in the
subjective night (E).The rhythm is entrained to a period of exactly 24 h in
appropriate zeitgebercycles of light and darkness (F). Entrainment to non-
24-h T cycles is also possible,as shown in (G), in which the phase settles later
in the short, 20-h cycle of warm and cold than it would in a 24-h cycle.If an
entraining cycle is about one-half of the free-running period, a frequency
demultiplication occurs,whereby one bout occurs per two cycles (H,
showing 12-h temperature cycles).
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many plants is triggered by day length. Entire animal reproductive
systems (from gene expression to anatomical structures) wax and
wane with the seasons. Clever experimental light-cycle protocols
have shown that the circadian system is at least part of the mecha-
nism that regulates photoperiodism (Saunders, 1990), thereby
increasing reproductive fitness (Beaver et al, 2002).
As the genetic era matures, so does the availability of and
knowledge about clock mutants. Recent reports indicate that
clock mutant animals can suffer from pathologies in addition to
their aberrant circadian behaviour. Increased incidence of radia-
tion-induced cancers occur in Period 2 (Per2) mutant mice (Fu
et al, 2002), and Clock mutants tend to develop obesity (Turek
et al, 2005). Whether and how these pathologies are related to
defective clock functions is not known, but lack of coordinated
metabolic regulation might be part of the cause. When construct-
ing a network of feedback loops in computer simulation, it is quite
difficult to establish conditions that prevent chaotic behaviour
(Roenneberg & Merrow, 2002)—these special conditions seem to
have evolved in the form of the circadian clock. Among them is
not only endogenous rhythmicity but also conditional responsive-
ness to environmental stimuli, which is necessary to prevent
chaotic behaviour.
The genes in the clock
Practically all of the success stories in deciphering a biological
mechanism include genetic approaches. Furthermore, numbers
are the geneticist’s best friend, and the circadian system offers
highly quantifiable traits: period length in constant conditions
and phase in entrainment. A genetic basis for the free-running cir-
cadian rhythm was suggested as early as 1932, by breeding bean
plants and selecting for subpopulations with short or long periods
(Bünning, 1932). The clock’s complex genetic nature could
already have been predicted by the bell-shaped distribution of the
progeny, obtained from crossing long- and short-period individu-
als. Numerous examples of cellular circadian systems argued that
the clock is not simply an emergent property of a complex (multi-
organ) system but is based within individual cells. The complexity
of the clock was viewed as an obstacle to its genetic analysis.
Eventually, pioneering mutagenesis screens revealed the first
clock gene (Period) in Drosophila melanogaster (Konopka &
Benzer, 1971), followed by the frequency gene in N. crassa
(Feldman & Hoyle, 1973). Many clock genes have since been
identified in these and other model systems—for example, mice,
Xenopus, Arabidopsis and Synechococcus.
Clock proteins in animals, plants, fungi and bacteria are largely
unrelated by sequence. However, similar molecular mechanisms
are apparent: clock genes and their proteins form a transcrip-
tion–translation regulatory loop with positive and negative feed-
back controls (Young & Kay, 2001). As a result, many clock genes
and clock-controlled genes—pure outputs of the clock so that
their mutations do not affect rhythm generation—cycle with a
coordinated period throughout the organism, both in vivo (Akhtar
et al, 2001; Storch et al, 2002), as well as in culture (Balsalobre
et al, 1998).
Other clock components, such as kinases or transcription factors
that drive the feedback loop, are constitutively expressed. Post-tran-
scriptional and post-translational modifications (predominantly phos-
phorylation so far) are also key regulators in the clock mechanism.
Phosphorylation is generally thought to regulate the clock by modulat-
ing clock-protein half-life (Görl et al, 2001; Liu et al, 2000; Young &
Kay, 2001), but it could also regulate subcellular compartmental shut-
tling or the activation/inactivation of transcription factor activity.
?
Fig 2 | The circadian system in animals is organized hierarchically. Molecular oscillations are generated at the cellular level,in which clock components include
transcription–translation feedback loops and possibly metabolic regulatory pathways (left).Organ or peripheral clocks develop a coordinated rhythm that is
synchronized relative to a pacemaker in the brain. The most obvious manifestation of this timing system is the sleep–wake cycle,but hundreds of parameters—
from cognitive functions to circulating hormone levels—are also changing over the 24-h day. Coupling between the brain and oscillators in the periphery has been
shown, and it can be disrupted (Damiola et al,2000), but feedback from peripheral clocks to the brain has yet to be formally demonstrated.
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Putative clock genes (identified through mutagenesis, interac-
tion and homology) are typically probed with reverse genetic
approaches, in which candidates are knocked out and the resultant
mutants assayed for clock properties. Co-regulatory effects on the
expression of other clock genes are used to establish how a gene
functions in the clockwork. For example, expression levels of
Cryptochromes 1 (Cry1) and 2 (Cry2) in Clock mutant mice are
non-rhythmic and low, indicating that these components of the
transcription–translation feedback loop are downstream of the
activator complex formed by the Clock and Brain and Muscle Arnt-
like protein 1 (BMAL1; Kume et al, 1999). Molecular clock models
have accordingly become an extensive network of interlocking
loops, without revision of the basic feedback loop hypothesis.
A recent report may help to promote a re-assessment of this
view: circadian oscillations have been reconstituted in a test tube
using just three Synechococcus proteins together with ATP
(Nakajima et al, 2005). The test tube oscillation is temperature-
compensated with little apparent damping and even adopts
appropriate periods when mutant proteins are added. The mea-
sured output was the phosphorylation of the cyanobacterial clock
protein KaiC in the presence of its modifiers KaiA and KaiB. The
authors concluded that this metabolic phosphorylation oscilla-
tor—not the transcriptional–translational feedback loop formed
by KaiA, KaiB and KaiC and their respective protein products—
generates the circadian rhythms, but that this could be a peculiari-
ty of a prokaryotic clock. However, similar to the readout in the
Synechococcus experiment, every molecular clock network
described so far has at least one component that is rhythmically
phosphorylated in a predictable way. Indeed, kinase mutants rep-
resent some of the most severe clock phenotypes (Lowrey et al,
2000; Price et al, 1998).
How might a metabolic oscillator be consistent with the nega-
tive feedback mechanism involving transcription and translation?
In a single organism, two oscillators can run free almost indepen-
dently of each other. This has been dramatically shown in temporal
isolation experiments in humans: the intervals between activity,
rest, meals, defaecation and even subjective estimations of when
an hour has passed can be stretched out to a circa 48-h (or longer)
day in these constant conditions. However, the period of the core
body temperature rhythm remains around 25 h (Aschoff et al,
1967). More recently, using jet-lag simulations in mice, the rhythm
of clock-gene expression was dissociated from that of neuronal
firing rate in the SCN pacemaker (Vansteensel et al, 2003).
Physiological experiments show that there are multiple oscilla-
tors even in single-cell and syncitial organisms: in the fungus
N. crassa or the dinoflagellate Gonyaulax polyedra, two oscilla-
tors can run independently under constant conditions (Correa et al,
2003; Roenneberg & Morse, 1993). Indications of mechanisms
beyond the transcription–translation feedback loop are not only
observed when circadian systems run free in constant conditions.
In Neurospora, it is now broadly acknowledged that frequency
(frq)-less Neurospora strains are systematically entrained by tem-
perature cycles (Merrow et al, 1999), and self-sustained rhythms
persist under some conditions in clock mutant strains (Lakin-
Thomas & Brody, 2000). In a wild-type strain, entrained phase in
Neurospora correlates with clock protein levels, but not necessar-
ily with the respective RNA expression (Tan et al, 2004), which
suggests uncoupling of the feedback loop (although not all of its
components) from clock function. A coupling ‘agent’ might be
suggested by work showing that clock transcription factor activity
can be regulated by the redox state of the cell (Rutter et al, 2001).
A synthesis of these findings indicates a conceptual framework
that incorporates at least two main oscillatory systems (Fig 2, left),
one stringently based on rhythmic transcription, the other appar-
ently not. As coupling between oscillatory systems is altered (due
to photoperiod, nutrition, temperature, and so on), different com-
ponents in the system might become dominant over others. The
necessity to accommodate changing conditions or seasons might
explain the complexity of the circadian clock.
The clock in our genes
Clock genes in humans were revealed through their homology to
those in mice. However, at this time, there are no (published) cir-
cadian methods using human cells in tissue culture that would
facilitate functional clock studies. In humans, we rely on entrain-
ment to allow genetic studies. Entrained phase is described by
‘chronotype’, a term that reflects the preferred timing of activity
and rest during the day. Chronotype can be assessed with a simple
questionnaire asking about sleep and wake times on work and on
free days (Roenneberg et al, 2004). The chronotype distribution in
the population is almost normal, with the mean mid-sleep time on
free days at about 04:30. Few individuals lie at the extreme ends
(pronounced morning and evening types, larks and owls, respec-
tively): 0.2% of people are more than 3 h earlier than the mean
(that is, go to bed around 21:30 or earlier) and 4.5% prefer to
sleep 3 h later (that is, go to bed around 03:30 or later). The bell-
shaped distribution contrasts with discrete Mendelian inheritance
patterns, which suggests the involvement of many genes (or at
least many polymorphisms in several genes), making the search
for human clock genes more difficult.
But what does chronotype tell us about the circadian clock? The
relationship between free-running period and chronotype
(entrained phase) was established long ago, showing that organ-
isms with short free-running periods entrain earlier in identical
zeitgeber cycles compared with those that have long free-running
periods (Pittendrigh & Daan, 1976). Similarly, chronotype (or diur-
nal preference) and free-running periods show a highly significant
correlation in humans (Duffy et al, 2001). At least in one human
subject, a highly penetrant per2 allele confers early chronotype as
well as short period (Toh et al, 2001). In addition to a genetic dis-
position, chronotype changes with age, becoming progressively
later during adolescence and then gradually shifting earlier again
(Roenneberg et al, 2004).
We can start to understand and probe the strategy of using
chronotype to find human clock genes by studying the mouse
model system (despite the fact that mice are nocturnal). Mutant
mice have been generated for many putative clock genes, and
their activity rhythms have been recorded both in constant and
entrained conditions. One study has specifically and systemati-
cally addressed entrained phase in clock mutant mice (Spoelstra
et al, 2004). The differences in entrained phase (using traditional
12 h light:12 h dark cycles) are minimal in Cry1 and Cry2 mice
compared with background-matched wild-type controls (less than
0.2 h), despite large differences in free-running periods. The lack
of correspondence between phase and period in these mutant
animals still has to be scrutinized experimentally (for example,
by varying the zeitgeber strengths and lengths, which should
both alter entrained phase). Other mouse mutants do ‘obey’ the
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EMBO reports VOL 6 | NO 10 | 2005 ©2005 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION
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M.Merrow et al
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phase-period law. The free-running period of the Per1 Cry2 double
mutant is 30 min longer and becomes active about 4 h later in
light:dark cycles than that of the wild type (Oster et al, 2003).
Ironically, the same double mutant illustrates the inherent
complications of approaching human clock genes by using
chronotype as a clock phenotype. Mouse Per1 Cry2 mutants
result from a cross between a short period (Per1, 1 h shorter than
wild type) and a long period (Cry2, 0.8 h longer) mutant. The
entrained phase of Per1 complies with the phase-period rule
(0.4 h earlier), whereas the chronotype of Cry2 is like that of wild
type. These results show that neither phase nor period is inherited
in a simple and predictable manner. Investigations into how this
works will help to genetically characterize the bell-shaped
chronotype distribution.
As our society moves towards a worldwide ‘24/7’ culture, with
shift work and jet lag almost the norm, circadian clock research is
becoming highly relevant to human health, behaviour and quality
of life. Chronotype is our only handle on human clock genetics in
real life. Understanding the complex genetics (and possibly epi-
genetics) in laboratory-reared mice, Arabidopsis, Neurospora and
Synechococcus will also help in the understanding of the rich
variation in circadian clocks in humans, who—besides inheriting
certain clock properties—are exposed to seasons, cultures, social
pressures and age-related chronotype changes.
ACKNOWLEDGEMENTS
We thank S. Daan for comments.Our work is supported by the Deutsche
Forschungsgemeinschaft, the Nederlandse Organisatie voor
Wetenschaappelijk Onderzoek,the European Commission and the Gottlieb
Daimler und Karl Benz-Stiftung.
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The circadian cycle
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CYCLES REVIEW SERIES
See other issues of EMBO reports for the complete Cycles Review Series:
Organisms
The circadian cycle
by Martha Merrow, Kamiel Spoelstra & Till Roenneberg
Cells
The chromosome cycle
by J. Julian Blow & Tomoyuki U.Tanaka
Receptors
GLUT4 recycling
by Chandrasagar B. Dugani & Amira Klip
Molecules
Cycling in ATPase
by Peter Dimroth
Martha Merrow,
Kamiel Spoelstra (top right)
& Till Roenneberg
M
2n
S
4n
GLUT4
