J R Soc Med 2003;96:486-489
doi:10.1258/jrsm.96.10.486
© 2003 Royal Society of Medicine
Clocks, genes and sleep
Malcolm von Schantz PhD
Simon N Archer PhD
Centre for Chronobiology, School of Biomedical and Molecular Sciences,
University of Surrey, Guildford GU2 7XH, UK
Correspondence to: M von Schantz E-mail:
m.von.schantz{at}surrey.ac.uk
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INTRODUCTION
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Do but consider what an excellent thing sleep is; it is so inestimable a
jewel that, if a tyrant would give his crown for an hour's slumber, it cannot
be bought; of so beautiful a shape is it, that though a man lie with an
Empress, his heart cannot beat quiet till he leaves her embracements to be at
rest with the other: yea, so greatly indebted are we to this kinsman of death,
that we owe the better tributary, half of our life to him: and there is good
cause why we should do so: for sleep is that golden chain that ties health and
our bodies together.—Thomas Dekker (The Gull's Hornbook,
1609)
Circadian rhythms are endogenously generated rhythms that approximate the
length of a day and a night. They are found in most organisms whose life span
is counted in days or greater units, rather than
hours.1 They enable
the organism to anticipate, rather than passively adjust to, the changes
imposed by alternation between day and night. These changes relate to positive
functions such as vision and negative effects such as ultraviolet light
damage. They also include indirectly generated alternations such as
temperature, availability of food and prevalence of predators.
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PHYSIOLOGY
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In mammals, circadian rhythms are governed by a paired master
oscillator in
the suprachiasmatic nuclei (SCN), which controls
slave oscillators throughout
the
body.
2,3
The SCN determines
the individual's free-running circadian period,
,
4 which in
man
is reported to average 24.2 h in the absence of light
cues.
5 Our
laboratory has observed a range of 23.83-25.00 h in blind
individuals.
6
Variability within the species, whereby individuals
reach peak performance at
different times of day, may have
conveyed an evolutionary advantage to early
human societies.
In a normal individual the phase of the SCN oscillator is
entrained
each day to the external light/dark cycle by information relayed
through the retinohypothalamic
tract.
7,8
In vertebrates, sleep has evolved as a sustained period of physical
inactivity during a specific part of the circadian
cycle.9 The
distribution of rest and activity between the night and the day varies between
taxa. Our reptilian ancestors, being exothermic, were active during the day
(diurnal), but the endothermic mammals that radiated from these species were
nocturnal. From there, various mammals (including most primates) reverted to
diurnality through secondary modifications, notably in the visual system. The
retina was recolonized by colour-sensitive cones (not required for night
vision) and the higher visual centres were
rewired.9-13
The parameters within the circadian system that define nocturnality and
diurnality are only partly
understood.14 As a
general rule, nocturnal species have a <24 hours, diurnal species have
a >24
hours.15 In
addition, the composition of visual photoreceptors seems important. People who
are colour blind through complete lack of cone photoreceptors tend to be
photophobic, and on the Micronesian island of Pingelap, where the condition is
common, some affected individuals specialize in night
fishing.16
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GENES AND MOLECULES
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Information on how circadian rhythms are generated at molecular
level comes
mainly from studies in mice. The mechanism depends
on tightly controlled
concerted coexpression of specific clock
genes. Most of these genes are
remarkably conserved amongst
coelomates
17—including
insects, molluscs
18
and vertebrates—although
the precise roles of specific components have
drifted during
evolution. At the centre of the machinery in mammals are the
Period (
Per1, Per2 and
Per3) and
Cryptochrome (
Cry1 and
Cry2)
genes. The protein
products of all these oscillate over the
24-hour cycle by inhibiting their own
promoters operating in
an intricate negative feedback loop.
Permanently attached to these promoters is a heterodimer of two
transcriptional activator proteins, CLOCK and
BMAL1.19 The
undisturbed activation of the Per and Cry promoters leads to
the accumulation of Per and Cry mRNA, which in the
suprachiasmatic nucleus takes place during the latter part of the day and the
early part of the night. Exported to the cytoplasm, these mRNA transcripts are
translated into the proteins PER1, PER2 and PER3 and CRY1 and CRY2, the
concentrations of which peak during the middle of the night. The production of
sufficient quantities of these proteins allows the formation of trimeric
complexes between CRY, PER and an additional clock component, casein kinase I
(CK I). All combinations of the different PER and CRY types seem to be
possible; in addition, the two CK I forms 
and 
are apparently
interchangeable. This trimeric complex enters the suprachiasmatic nucleus,
where it binds to the CLOCK:BMAL1 complex and prevents its positive effect on
the transcription of Per and Cry
mRNA.3
Phosphorylation by casein kinase I appears important both in targeting clock
gene proteins for transport into the nucleus and in their eventual
degradation. This prevents PER and CRY protein from acting on their own
promoters, and their temporary absence once again allows the unperturbed
action of BMAL1 and CLOCK on the Per and Cry promoters,
starting the whole cycle anew. An added twist to this negative feedback loop
is a positive one; the PER 2 protein has been shown to be a positive regulator
of BMAL1 gene expression, which also cycles whilst the level of its
partner CLOCK remains
constant.20
Clearly the mechanisms that regulate each person's with such
precision are complex (Figure
1), so it is not surprising that mutations in the different clock
genes can affect the circadian output. The Clock gene was identified
in a mouse strain with a long and unstable circadian
period,21 and
knockout mice have been produced lacking the different Per and
Cry genes and combinations
thereof.3 Whereas
single knockouts of either one of the Per or Cry genes will
alter the circadian period, functional double knockouts of Per1 and
Per222 and
of Cry1 and
Cry223
abolish circadian rhythmicity altogether. The effect of perturbing the
expression of Per3 is only to shorten the circadian
period.24 A
shortened period is also observed in the mutant strain of the Syrian
golden hamster,25
in which a point mutation in casein kinase decreases its ability to
phosphorylate both positive and negative regulators of the circadian
mechanism.26,27
The net result is likely to be less degradation of the phosphorylated
protein.
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Figure 1. The 24-hour cycle of clock gene expression. (a) In the
morning, the promoters of the Cry and Per genes are
activated by the CLOCK and BMAL1 proteins, producing mRNA transcripts that are
exported into the cytoplasm. (b) During the day, ribosomes bind to
these mRNA molecules and translate their genetic code into proteins.
(c) In the latter part of the day, PER (1, 2 and 3) and CRY (1 and 2)
proteins accumulate in the cytoplasm, where CK I ( and ) are
present constitutively. (d) In the evening, CRY, PER, and CK I
molecules bind to each other in different combinations. PER is phosphorylated
by CK I, and the complexes enter the nucleus in a coordinated fashion at
night. (e) During the rest of the night, the activation of the
Cry and Per promoters by CLOCK:BMAL1 is inhibited by the
CRY:PER:CK I complex. As a result, production of Cry and Per
mRNA is interrupted, which in turn interrupts production of CRY and PER
proteins. (f) As both the mRNA and the proteins are short-lived, they
will gradually disappear towards the morning. The cycle is complete, and the
production of Cry and Per mRNA starts again
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SLEEP DISORDERS
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Sleep has famously been described as being ‘of the brain,
by the
brain, and for the
brain’.
28 Its
relation to
the circadian clock is less simple to describe. Some disorders
of
sleep are unrelated to circadian
rhythms
29; others
are
undoubtedly related to it, particularly the advanced and delayed
sleep
phase
syndromes.
30,31
In these conditions, sleep occurs
either abnormally early or abnormally late.
This could theoretically
be caused either by an abnormal
or by abnormal
timing of the
sleep phase within a circadian cycle of normal periodicity.
From what we can tell from their sequence features, their function in
in vitro studies, and the rather limited insights we possess from
ex vivo material, human clock genes behave in a very similar fashion
to murine clock genes. But our secondary reversion to a diurnal lifestyle
(which did not happen in the mouse) will have been accompanied by limited but
important changes in the clock gene machinery, and these should soon be
identifiable through comparative analysis of the respective genomes.
In addition, differences are seen between members of the human race.
Polymorphisms in clock genes can be related to circadian parameters, and the
most famous finding so far is a large family where advanced sleep phase
syndrome seems to be inherited as a single-gene
defect.32 The
condition manifests itself in this family with advanced melatonin,
temperature, and sleep/wake rhythms co-segregating with a missense mutation in
the Per2
gene.33 Because of
the non-homologous aminoacid substitution, the resulting PER2 protein is
phosphorylated less efficiently by casein kinase than the native one—an
observation that offers a satisfying mechanistic explanation for the
similarity between this phenotype and that of the hamster, whose
missense mutation in casein kinase I results in essentially the same
net effect.
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MORNING AND EVENING PREFERENCES
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It is a common observation that some individuals are most alert
in the
morning, others in the evening. These preferences can
be quantified by the
Horne-Östberg (HO) questionnaire,
in which a low score indicates evening
preference
34 and a
long
.
35
Katzenberg and
co-workers
36
studied the relation between
HO score and a single-nucleotide polymorphism
(SNP) at position
3111 in the
Clock gene transcript. Although this
locus does
not alter the aminoacid sequence of the CLOCK protein, it could
nonetheless be important for the stability and translatability
of the
transcript. Individuals homozygous or heterozygous for
the 3111C allele of
Clock were reported to have an HO score
average of two points higher
than 3111T homozygotes. Our laboratory
attempted to confirm this finding by a
four-pronged
approach.
6 First, we
sought a correlation between the
Clock3111 polymorphism
and extreme
HO score in a population of 484 individuals. Secondly,
we sought a correlation
with
as determined in blind subjects
with free-running circadian rhythm.
Thirdly, we compared the
prevalence of this polymorphism in patients with
delayed sleep
phase syndrome and normal controls. Fourthly, we investigated
the expression levels of a reporter gene construct carrying
the two different
forms of the relevant section of the 3'-UTR
of
Clock. In none
of these studies did we observe any significant
effect of this
polymorphism.
The gene associated with evening preference that has produced the most
interesting results to date in humans is Per3. In a Japanese
population, Ebisawa and
colleagues37
reported that a rare single-nucleotide polymorphism causing an aminoacid
substitution correlated with delayed sleep phase syndrome. Our laboratory has
studied a more dramatic genetic polymorphism, initially described but not
characterized in Ebisawa's paper, encoding an 18-aminoacid tandem repeat
sequence, of which humans have either four or five successive copies in each
of their Per3
alleles.38 By
comparing HO-characterized subjects whose scores were around the mean for
their gender and age group with those with extreme evening and morning
preference, we were able to distinguish an excess prevalence of the shorter
repeat allele in subjects with extreme evening preference. Extending the study
to a cohort of patients with delayed sleep phase syndrome, we showed that the
association with the shorter allele was even stronger in this population.
Thus, although no physiological studies have formally linked the extremes of
evening preference and/or long with delayed sleep phase syndrome, it
would appear from the convergence of their Per3 genotype that such a
study is not only worthwhile but long overdue.
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CONCLUSION
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In mice, single null mutations in clock genes seem to correlate
neatly with
circadian phenotype in a monogenic mendelian inheritance
pattern. In man, the
known human clock gene differences appear
merely to predict a greater or
lesser propensity. One reason
for this difference is that most of the mouse
models studied
so far have been engineered to abolish the function of a
specific
gene, rather than carrying a more or less altered form of it.
Another
is that laboratory rodent strains are highly inbred
and thus much more
homogeneous with respect to all other clock
genes. The human circadian
genotype, being a polygenic trait,
is more akin to a hand of cards. Most of us
will have a hand
containing average cards or a balanced mixture of high and
low
ones. Only the hands that contain predominantly low or high
cards will
stand out. The great majority of us function normally
with our allotted
circadian phenotype and its interaction with
our environment, much as we are
able to deal with other aspects
of our genetic inheritance. But the minority
who have been
dealt a dud hand of the clock genes card game deserve more
sympathy
and clinical help than they are often accorded.
Our culture trends to associate early waking with virtue and industry, and
late sleeping with vice and lassitude. Lack of conformity with this norm is
not always a matter of choice: clearly, some people are genetically programmed
for an extreme diurnal preference.
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