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Clock-Controlled Genes
J ÜRGEN A. R IPPERGER , U RS A LBRECHT
Department of Medicine, Division of Biochemistry,
University of Fribourg, Fribourg, Switzerland
Synonyms
Circadian output genes
Definition
Genes whose time-of-day specific expression is dependent on the circadian oscillator.
Characteristics
The mammalian circadian oscillator is based on
interconnected transcriptional and post-translational
feedback loops. In the negative limb, transcriptional
repressors of the ▶Cryptochrome (Cry) and ▶Period
(Per) family periodically modulate the activation
potential of the transcription factors Clock (or Npas2)
and Bmal1 (see ▶clock genes). By contrast, in the
positive limb the orphan nuclear hormone receptors of
the Rev-erb family repress transcription in the opposite
phase of the negative limb. Both limbs together interact
and govern oscillations of gene expression with
a ▶free-running ▶period length of about a day. Due
to the make-up of the circadian ▶oscillator as
transcriptional feedback loops, it is not surprising that
most of the direct output is hardwired to the clock
mechanism (Fig. 1).
A recent in vitro study combined with a systems
biological approach came to the conclusion that the
phase of circadian expression resulted from the
utilization of three different types of response elements:
▶E-box motifs as binding sites for Clock (or Npas2)
and Bmal1, RORE motifs as binding sites for Ror and
Rev-erb family members, and D-elements as binding
sites for PAR bZip factors [1].
To address the question of how many clockcontrolled genes exist, DNA microarray experiments
have been conducted using the site of the central
oscillator, the ▶Suprachiasmatic nucleus (▶SCN) in
the brain as tissue source [2]. The obtained data were
filtered afterwards to identify periodically expressed
genes. Surprisingly, about 5% of the steady-state
mRNA was rhythmically expressed with robust but
sometimes low amplitude. This is still a rough estimate
since the analysis of steady-state mRNA does not
account for mRNA stability and many low-level
cycling genes maybe eliminated artificially by the
filters employed for data mining. The rhythmic genes
identified included those for prohormone/neuropeptide
synthesis, processing, and degradation, thought to be
one of the main outputs of the SCN to govern the
circadian rhythmicity of mammals. Some of these
hormones, like ▶pituitary adenylate cyclase-activating
▶polypeptide 1 (▶PACAP) and arginine vasopressin
(AVP) had been known before as rhythmically expressed genes in the SCN. Another important set of
coordinately expressed genes contained enzymes
important for carbon source utilization and oxidative
phosphorylation in the mitochondria. The detailed
analysis of this pathway suggested a circadian rhythm
in the energy metabolism and redox state of SCN
neurons.
A major surprise was the relatively small overlap of
rhythmic transcripts between different tissues examined. In the study by Panda et al. [2], about 330
rhythmic transcripts specific for either the SCN region
in the brain, or the liver were found and there were only
28 overlapping transcripts, which included most core
oscillator components. Therefore, the output genes are
not only subject to circadian control of gene expression,
but also to tissue-specific control. At the moment, we
have much better insight into the circadian control than
the tissue-specific control of circadian output genes.
However, it appears that both components together are
necessary to orchestrate the expression of genes in
a manner optimal for a specialized tissue such as the
SCN. In the context of this essay we will focus on
known clock-controlled genes in the brain.
The first gene to be analyzed in great detail and
linked to the molecular oscillator was the arginine vasopressin gene ([3], Fig. 1). This hormone is synthesized
mainly in the vasopressinergic neurons of the paraventricular nuclei (PVN) and the Supraoptical nuclei
(SON). It is released into the bloodstream from the
posterior pituitary to regulate the salt and water balance.
However, this hormone acts also as a neuropeptide
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Clock-Controlled Genes
Clock-Controlled Genes. Figure 1 Clock-controlled genes are linked to the circadian oscillator. Circadian output
genes are linked to the oscillator by E boxes, RORE, and/or D-elements. Per1 inhibits the activity of the Clock (C) or
Npas2 (N) and Bmal1 (B) heterodimers. Lactate dehydrogenase A (Ldha) can affect the redox state of a cell,
pydridoxal kinase (Pdxk) generates pyridoxal phosphate, a coenzyme involved in neurotransmitter synthesis, and
arginine vasopressin (Avp) can bind rhythmically to its receptor V1a on SCN neurons. For details, see text.
in the central nervous system (CNS). For instance, it is
rhythmically produced by the neurons of the SCN and
modulates the firing rate of SCN neurons in a very local
fashion. The expression of this gene was nearly
abolished in the SCN but not in the SON of
ClockΔ19/ClockΔ19 homozygous mutant mice, which
carry a dominant-negative version of the Clock protein.
Subsequent analysis revealed the importance of the
E-box motif in the promoter region of this gene as
a binding site for Clock and Bmal1. Altogether, the
data suggested that the arginine vasopressin gene was
directly hardwired to the molecular oscillator via its
E-box motif and the transcriptional activators Clock and
Bmal1. Interestingly, in the SON region there were
barely detectable levels of Bmal1, and this may impair
an effect of the ClockΔ19 mutation on the transcription
of this gene. However, it is still an unresolved issue,
why the expression of this gene is so highly specific for
the CNS.
The transcription factor D-site Binding Protein
(DBP) was originally thought to manifest a liverspecific regulator of the albumin gene. However, it was
identified as a ubiquitous output gene expressed with
very high circadian amplitude. Mice deficient of this
gene display a 30 min shorter free-running period
length indicating a feedback of this protein to the
circadian oscillator. As a rhythmically expressed
transcription factor, Dbp can amplify the action of the
circadian oscillator on many D-element bearing target
genes (Fig. 1). In triple knock out mice with an
inactivation of the Dbp gene and the two other members
of the PAR bZip transcription factors, sporadic and
audiogenic epileptic seizures occurred [4]. This phenotype was linked to a slight deregulation of the gene for
pyridoxal kinase (Pdxk), involved in the pathway for
the conversion of vitamin B6 derivates into pyridoxal
phosphate. Pyridoxal phosphate is a coenzyme of many
enzymes involved in the metabolism of various
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Clock-Controlled Genes
neurotransmitters. This is an example of a very drastic
phenotype provoked by a subtle deregulation of an
enzymatic activity. Thus, subtle circadian changes in
enzymatic activities can have a drastic influence on
physiology and metabolism.
The circadian regulation of the Dbp gene was
analyzed in great detail in the liver but the mechanism
is probably very similar for the SCN ([5], Fig. 2).
The transcription cycle of Dbp is initiated by binding
of Clock and Bmal1 to three defined E-box containing
regions within the gene. The binding of these factors
provokes a change in the local chromatin structure as
evidenced by the acetylation of lysine 9 of histone H3,
the trimethylation of lysine 4 of histone H3, and
a reduction of the histone density overall. Under these
conditions transcription of the gene commences. After
a certain time, Clock and Bmal1 fall off their target sites
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within Dbp, the transcription ceases, and the chromatin
closes in to form a heterochromatin-like, inactive
state. Upon re-binding of Clock and Bmal1 the next
day, another circadian cycle can start. While Dbp is
expressed in the liver and the SCN neurons with very
high circadian amplitude, its amplitude of cycling in the
other parts of the brain is much lower, indicating some
additional tissue-specific component.
Npas2 is an analog of Clock expressed mainly in
the forebrain. To address the regulatory potential of
this transcription factor, an inducible neuroblastoma
cell line for Npas2 and Bmal1 was engineered. One
surprising target gene upregulated after the induction
of these transcriptional regulators was the A isoform of
lactate dehydrogenase (Ldha) ([6], Fig. 1). This
enzyme reversibly catalyses the dehydrogenation of
pyruvate to lactate. Therefore, it has a direct impact on
Clock-Controlled Genes. Figure 2 Rhythmic binding of Clock (yellow) and Bmal1 (orange) to DNA governs
circadian Dbp transcription and chromatin transitions. During active transcription there are less histones (light blue
discs) around the promoter (red rectangle), and they are marked by acetylation of lysine 9 (blue stars) of histone H3
and trimethylation of lysine 4 (teal hexagons) of histone H3. During repression, the heterochromatin closes as
a consequence of change in methylation (pink hexagons), binding of heterochromatin binding protein 1 (HP1, rose
circles) and loss of acetylation of histones. The promoter (red) is packed by histones (blue disc) and the transcription
shuts off. The chromatin transitions and the transcription are dependent on rhythmic Clock and Bmal1 binding. Light
yellow background represents activation during the day, whereas blue shows repression during the night.
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Clock-Controlled Genes
the redox state within a cell by influencing the ratio
of reduced nicotinamide adenine dinucleotide (NADH)
to its oxidized form, NAD. Astonishingly, the heterodimer formation between Npas2 and Bmal1, and the
binding activity of this heterodimer to DNA were both
dependent on the ratio of NADH to NAD: the reduced
form repressed heterodimer formation and concomitantly DNA binding of the heterodimer. This led to an
interesting but still preliminary model of entrainment of
neurons by changes in the redox state of neurons: in
a first step, astrocytes take up extra-cellular glutamate
from the synaptic clefts secreted during neuronal
activity. This stimulates glycolysis in these cells and
subsequently the secretion of lactate. The lactate is
taken up by the neurons again and provokes circadian
fluctuations of the redox state that govern the activity of
the NPAS2 and BMAL1 heterodimers. This may in turn
rhythmically affect the neuronal activity and therefore
the periodic secretion of glutamate.
After these examples of clock-controlled genes that
are direct targets of activation by Clock (or Npas2) and
Bmal1, we now turn towards genes that contain binding
sites for the transcriptional repressors of the Rev-erb
family (Fig. 1). The function of ROREs within the
circadian clock was found by two independent
approaches: (i) the circadian amplitude of the expression of Bmal1 in the SCN and liver of ▶Rev-Erbα
homozygous knock out mice was severely dampened
[7]; (ii) in a DNA microarray study the circadian
transcripts of SCN and liver were grouped according to
their phases of expression, then the transcription start
sites were identified, and the promoter regions
subsequently analyzed for common binding motifs for
transcriptional regulators [8]. The target genes identified in this fashion included Bmal1, E4bp4, and the
arginine vasopressin receptor 1A (V1a). Interestingly,
E4bp4 is a repressor of transcription with the same
DNA binding specificity as the PAR bZip transcription
factors that become expressed in the opposite phase. It
is tempting to speculate that a particular target gene can
alternatively bind PAR bZip transcription factors or the
repressor E4bp4, allowing precise transcriptional
regulation. Circadian expression of the V1a receptor
was also an interesting finding since its ligand arginine
vasopressin is expressed in a different phase in the SCN
neurons (see above).
A completely different kind of circadian regulation is
found in the ▶pineal gland regarding the translation
of the rate-limiting enzyme in melatonin synthesis,
the arylalkylamine N-acetyltransferase (AANAT) ([9],
Fig. 3).
In rodents, the peaks of mRNA and protein accumulation are separated by four to six hours. This delay
is due to a co-translational regulatory mechanism.
Clock-Controlled Genes. Figure 3 Co-translational regulation of the arylalkylamine N-acetyltransferase (AANAT)
gene in the pineal gland important for melatonin production. High amounts of HnRNP Q protein are necessary to bind
to an IRES sequence within the 5′-untranslated region of the AANAT mRNA to allow for the formation of active
translation complexes to initiate the production of AANAT protein. Upon phosphorylation (p-AANAT) serotonin is
converted to melatonin. Light yellow background represents repression during the day, whereas blue shows
activation during the night.
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Clock-Controlled Genes
The 5′-untranslated region of the mRNA contained an
internal ribosome entry site (IRES), which allowed 5′Cap independent translation. However, this particular
IRES permitted the translation of the mRNA only in
the presence of sufficient amounts of Heterogeneous
nuclear ribonucleoprotein Q (hnRNP Q). This protein
bound specifically to the IRES sequence of AANAT and
recruited the ribosome complex to initiate the translation. HnRNP Q accumulated in the nuclei of pinealocytes in a circadian fashion, gating the translation of
the AANAT mRNA to a specific time-window. It is
tempting to speculate that this complicated mechanism
of co-translational regulation is a rather widespread
phenomenon within the mammalian circadian oscillator.
Taken together, clock-controlled genes within the
CNS and other tissues are controlled by different
mechanisms. The simplest fashion is a direct coupling
of the target genes to the core oscillator via Clock (or
Npas2) and Bmal1, or the Rev-erb family. A more
indirect way exploits various transcriptional regulators,
e.g., Dbp and E4bp4, as intermediaries. Maybe this
could account also for the differences observed in the
circadian clocks of different tissues. One should keep
also in mind that changes in the transcriptional status of
a gene not necessarily reflect drastic changes in the
protein levels, and vice versa (see the co-translational
mechanism).
Where does the research go? Many mental syndromes like depression, mania, and bipolar disorder are
somehow linked to the circadian clock. Therefore, it is
an important task to identify potential target genes
whose unbalanced circadian expression interferes with
the normal health status. However, this task is by far not
an easy one, since even subtle changes in the level of
neurotransmitters might have drastic effects as seen, for
example, for the pyridoxal kinase. Another example
concerns the influence of the clock gene Per2 on
alcohol consumption [10]. In Per2 Brdm1 homozygous
mutant mice, the expression of an astrocyte-specific
glutamate transporter is slightly down regulated,
provoking a hyper-glutamatergic state within the
CNS. As a consequence, the animals consume more
and are more resistant to alcohol. The effect of the Per2
mutation can be reverted by the application of
acamprostate, a drug that is thought to act by
dampening a hyper-glutamatergic state. As an estimate,
5
10% of alcoholic patients respond well to this drug. In
the future, with a more detailed knowledge of the
circadian oscillator of the CNS and its target genes, it
will be possible to understand and to develop new
therapies that will help to treat mental disorders.
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