CLOCK
Encyclopedia
Clock is a gene encoding a basic-helix-loop-helix
-PAS transcription factor
(CLOCK) that affects both the persistence and period of circadian rhythms. CLOCK functions as an essential activator
of downstream elements in the pathway critical to the generation of circadian rhythms.
and his colleagues. Takahashi used forward mutagenesis screening of mice treated with N-ethyl-N-nitrosourea
to create and identify mutations in key genes that broadly affect circadian activity. The Clock mutants discovered through the screen displayed an abnormally long period of daily activity. This trait proved to be heritable, and mice bred to be homozygous for the mutation eventually lost all circadian rhythmicity after several days in constant darkness. This showed that intact Clock genes are necessary for normal mammalian circadian function.
CLOCK protein has been found to play a central role as a transcription factor in the circadian pacemaker. In Drosophila
, newly synthesized CLOCK (CLK) is hypophosphorylated in the cytoplasm before entering the nucleus. Once in the nuclei, CLK is localized in nulear foci and is later redistributed homogeneously. CYCLE(CYC) (also known as dBMAL for the BMAL1 ortholog in mammals) dimerizes with CLK via their respective PAS domain
s. This dimer then recruits co-activator CREB-binding protein(CBP) and is further phosphorylated. Once phosphorylated, this CLK-CYC complex binds to the E-box
elements of the promoters of period (per) and timeless
(tim) via its bHLH domain, causing the stimulation of gene expression of per and tim. A large molar excess of period (PER) and timeless (TIM) proteins causes formation of the PER-TIM heterodimer which prevents the CLK-CYC heterodimer from binding to the E-boxes of per and tim, essentially blocking per and tim transcription. CLK is hyperphosphorylated when double-time (DBT) kinase
interacts with the CLK-CYC complex in a PER reliant manner, destabilizing both CLK and PER, leading to the degradation of both proteins.
Hypophosphorylated CLK then accumulates, binds to the E-boxes of per and tim and activates their transcription once again. This cycle of post-translational phosphorylation suggest that temporal phosphorylation of CLK helps in the timing mechanism of the circadian clock.
A similar model is found in mice, in which BMAL1 dimerizes with CLOCK to activate per and cryptochrome
(cry) transcription. PER and CRY proteins form a heterodimer which acts on the CLOCK-BMAL heterodimer to repress the transcription of per and cry.
CLOCK exhibits histone acetyl transferase (HAT) activity, which is enhanced by dimerization with BMAL1. Dr. Paolo Sassone-Corsi and colleagues demonstrated in vitro
that CLOCK mediated HAT activity is necessary to rescue circadian rhythms in Clock mutants.
allele at the Clock locus that codes for an antagonist to the wild-type protein. The presence of an antimorphic protein downregulates the transcriptional products normally upregulated by Clock.
that eliminates the activation domain of the CLOCK protein, causes dominant effects: half of the heterozygous flies with this mutant gene have an altered period while the other half become arrhythmic. Homozygous flies are all arrhythmic. Furthermore, these mutant flies express low levels of PER and TIM proteins, indicating that Clock functions as a positive element in the circadian loop.
to the Jrk mutant is the ClockΔ19 mutant that possesses a deletion in exon
19 of the Clock gene. This dominant-negative mutation results in a defective CLOCK-BMAL dimer, which causes mice to have a decreased ability to activate per transcription. In constant darkness, ClockΔ19 mice heterozygous for the Clock mutant allele exhibit lengthened circadian periods, while ClockΔ19/Δ19 mice homozygous for the allele become arrhythmic. In both heterozygotes and homozygotes, this mutation also produces lengthened periods and arrhythmicity at the single-cell level.
Clock -/- null mutant mice, in which Clock has been knocked out, display completely normal circadian rhythms. The discovery of a null Clock mutant with a wild-type phenotype directly challenged the widely accepted premise that Clock is necessary for normal circadian function. Furthermore, it suggested that the CLOCK-BMAL1 dimer need not exist to modulate other elements of the circadian pathway. Neuronal PAS domain
containing protein 2 (NPAS2
, a CLOCK analog
) can substitute for CLOCK in these Clock-null mice.
The CLOCK-BMAL dimer acts as a positive limb of a feedback loop. The binding of CLOCK-BMAL to an E-box promoter element activates transcription of clock genes such as per1, 2, and 3 and tim in mice. It has been shown in mice that CLOCK-BMAL also activates the Nicotinamide phosphoribosyltransferase
gene (also called Nampt), part of a separate feedback loop. This feedback loops creates a metabolic oscillator. The CLOCK-BMAL dimer activates transcription of the Nampt gene, which codes for the NAMPT protein. NAMPT is part of a series of enzymatic reactions that covert niacin
(also called nicotinamide
) to NAD
. SIRT1, which requires NAD for its enzymatic activity, then uses increased NAD levels to deacetylate BMAL1, suppressing it. This suppression results in less transcription of the NAMPT, less NAMPT protein, less NAD made, and therefore less SIRT1 and less suppression of the CLOCK-BMAL dimer. This dimer can again positively activate the Nampt gene transcription and the cycle continues, creating another oscillatory loop involving CLOCK-BMAL as positive elements. The key role that Clock plays in metabolic and circadian loops highlights the close relationship between metabolism and circadian clocks.
in Clock, rs6832769, may be related to the personality trait agreeableness
. Another single nucleotide polymorphism
(SNP) in Clock, 3111C, has been associated with diurnal preference. This SNP is also associated with increased insomnia
, difficulty losing weight, and recurrence of major depressive episodes in patients with bipolar disorder
.
In mice, Clock has been implicated in sleep disorders, metabolism
, pregnancy
, and mood disorders. Clock mutant mice sleep less than normal mice each day. The mice also display altered levels of plasma glucose
and rhythms in food intake. These mutants develop metabolic syndrome
symptoms over time. Furthermore, Clock mutants demonstrate disrupted estrous cycle
s and increased rates of full-term pregnancy failure. Mutant Clock has also been linked to bipolar disorder
-like symptoms in mice, including mania
and euphoria
. Clock mutant mice also exhibit increased excitability of dopamine
neurons in reward centers of the brain. These results have led Dr. Colleen McClung to propose using Clock mutant mice as a model for human mood and behavior disorders.
The CLOCK-BMAL dimer has also been shown to activate reverse-erb receptor alpha (Rev-ErbA alpha
) and retinoic acid orphan receptor alpha (ROR-alpha). REV-ERBα and RORα regulate Bmal by binding to retinoic acid-related orphan receptor response elements (ROREs) in its promoter.
In 2010, a Yale University team led by Dr. Yong Zhu found that variations in the epigenetics
of the Clock gene may lead to an increased risk of breast cancer. It was found that in women with breast cancer, there was significantly less methylation of the Clock promoter region. It was also noted that this effect was greater in women with estrogen and progesterone receptor-negative tumors.
The CLOCK gene may also be a target for somatic mutations in microsatellite unstable colorectal cancers. In a study done in 2010 by researchers in the University of Helsinki, 53% of putative novel microsatellite instability target genes responsible for colorectal cancer contained CLOCK mutations.
Basic-helix-loop-helix
A basic helix-loop-helix is a protein structural motif that characterizes a family of transcription factors.- Structure :The motif is characterized by two α-helices connected by a loop. In general, transcription factors including this domain are dimeric, each with one helix containing basic amino...
-PAS transcription factor
Transcription factor
In molecular biology and genetics, a transcription factor is a protein that binds to specific DNA sequences, thereby controlling the flow of genetic information from DNA to mRNA...
(CLOCK) that affects both the persistence and period of circadian rhythms. CLOCK functions as an essential activator
Activator (genetics)
An activator is a DNA-binding protein that regulates one or more genes by increasing the rate of transcription. The activator may increase transcription by virtue of a connected domain which assists in the formation of the RNA polymerase holoenzyme, or may operate through a coactivator. A...
of downstream elements in the pathway critical to the generation of circadian rhythms.
Discovery and function
The Clock gene was first identified in 1994 by Dr. Joseph TakahashiJoseph Takahashi
Joseph S. Takahashi is a Japanese American neurobiologist and geneticist. Takahashi is a professor at University of Texas Southwestern Medical Center as well as an investigator at the Howard Hughes Medical Institute. Takahashi's research group discovered the genetic basis for the mammalian...
and his colleagues. Takahashi used forward mutagenesis screening of mice treated with N-ethyl-N-nitrosourea
ENU
ENU, also known as N-ethyl-N-nitrosourea , is a highly potent mutagen. For a given gene in mice, ENU can induce 1 new mutation in every 700 loci. It is also toxic at high doses....
to create and identify mutations in key genes that broadly affect circadian activity. The Clock mutants discovered through the screen displayed an abnormally long period of daily activity. This trait proved to be heritable, and mice bred to be homozygous for the mutation eventually lost all circadian rhythmicity after several days in constant darkness. This showed that intact Clock genes are necessary for normal mammalian circadian function.
CLOCK protein has been found to play a central role as a transcription factor in the circadian pacemaker. In Drosophila
Drosophila
Drosophila is a genus of small flies, belonging to the family Drosophilidae, whose members are often called "fruit flies" or more appropriately pomace flies, vinegar flies, or wine flies, a reference to the characteristic of many species to linger around overripe or rotting fruit...
, newly synthesized CLOCK (CLK) is hypophosphorylated in the cytoplasm before entering the nucleus. Once in the nuclei, CLK is localized in nulear foci and is later redistributed homogeneously. CYCLE(CYC) (also known as dBMAL for the BMAL1 ortholog in mammals) dimerizes with CLK via their respective PAS domain
PAS domain
The PAS domain is a protein domain contained in many signaling proteins where it functions as a signal sensor. PAS domains are found in a large number of organisms from bacteria to humans...
s. This dimer then recruits co-activator CREB-binding protein(CBP) and is further phosphorylated. Once phosphorylated, this CLK-CYC complex binds to the E-box
E-box
An E-box is a DNA sequence which usually lies upstream of a gene in a promoter region. It is a transcription factor binding site where the specific sequence of DNA, CANNTG, is recognized by proteins that can bind to it to help initiate its transcription. Once transcription factors bind to...
elements of the promoters of period (per) and timeless
Timeless (gene)
Timeless is a gene in Drosophila which encodes TIM, an essential protein that regulates circadian rhythms. Timeless mRNA and protein oscillate rhythmically with time as part of a transcription-translation [negative feedback] loop involving the period gene and its protein.-Discovery:Timeless was...
(tim) via its bHLH domain, causing the stimulation of gene expression of per and tim. A large molar excess of period (PER) and timeless (TIM) proteins causes formation of the PER-TIM heterodimer which prevents the CLK-CYC heterodimer from binding to the E-boxes of per and tim, essentially blocking per and tim transcription. CLK is hyperphosphorylated when double-time (DBT) kinase
Protein kinase
A protein kinase is a kinase enzyme that modifies other proteins by chemically adding phosphate groups to them . Phosphorylation usually results in a functional change of the target protein by changing enzyme activity, cellular location, or association with other proteins...
interacts with the CLK-CYC complex in a PER reliant manner, destabilizing both CLK and PER, leading to the degradation of both proteins.
Hypophosphorylated CLK then accumulates, binds to the E-boxes of per and tim and activates their transcription once again. This cycle of post-translational phosphorylation suggest that temporal phosphorylation of CLK helps in the timing mechanism of the circadian clock.
A similar model is found in mice, in which BMAL1 dimerizes with CLOCK to activate per and cryptochrome
Cryptochrome
Cryptochromes are a class of blue light-sensitive flavoproteins found in plants and animals. Cryptochromes are involved in the circadian rhythms of plants and animals, and in the sensing of magnetic fields in a number of species...
(cry) transcription. PER and CRY proteins form a heterodimer which acts on the CLOCK-BMAL heterodimer to repress the transcription of per and cry.
CLOCK exhibits histone acetyl transferase (HAT) activity, which is enhanced by dimerization with BMAL1. Dr. Paolo Sassone-Corsi and colleagues demonstrated in vitro
In vitro
In vitro refers to studies in experimental biology that are conducted using components of an organism that have been isolated from their usual biological context in order to permit a more detailed or more convenient analysis than can be done with whole organisms. Colloquially, these experiments...
that CLOCK mediated HAT activity is necessary to rescue circadian rhythms in Clock mutants.
Mutants of Clock
Clock mutant organisms can either possess a null mutation or an antimorphicMuller's morphs
1946 Nobel Prize winner Hermann J. Muller coined the terms amorph, hypomorph, hypermorph, antimorph and neomorph to classify mutations based on their behaviour in various genetic situations. These classifications are still widely used in Drosophila genetics to describe mutations...
allele at the Clock locus that codes for an antagonist to the wild-type protein. The presence of an antimorphic protein downregulates the transcriptional products normally upregulated by Clock.
Drosophila
In Drosophila, a mutant form of Clock (Jrk) resulting from a premature stop codonStop codon
In the genetic code, a stop codon is a nucleotide triplet within messenger RNA that signals a termination of translation. Proteins are based on polypeptides, which are unique sequences of amino acids. Most codons in messenger RNA correspond to the addition of an amino acid to a growing polypeptide...
that eliminates the activation domain of the CLOCK protein, causes dominant effects: half of the heterozygous flies with this mutant gene have an altered period while the other half become arrhythmic. Homozygous flies are all arrhythmic. Furthermore, these mutant flies express low levels of PER and TIM proteins, indicating that Clock functions as a positive element in the circadian loop.
Mice
The mouse homologHomology (biology)
Homology forms the basis of organization for comparative biology. In 1843, Richard Owen defined homology as "the same organ in different animals under every variety of form and function". Organs as different as a bat's wing, a seal's flipper, a cat's paw and a human hand have a common underlying...
to the Jrk mutant is the ClockΔ19 mutant that possesses a deletion in exon
Exon
An exon is a nucleic acid sequence that is represented in the mature form of an RNA molecule either after portions of a precursor RNA have been removed by cis-splicing or when two or more precursor RNA molecules have been ligated by trans-splicing. The mature RNA molecule can be a messenger RNA...
19 of the Clock gene. This dominant-negative mutation results in a defective CLOCK-BMAL dimer, which causes mice to have a decreased ability to activate per transcription. In constant darkness, ClockΔ19 mice heterozygous for the Clock mutant allele exhibit lengthened circadian periods, while ClockΔ19/Δ19 mice homozygous for the allele become arrhythmic. In both heterozygotes and homozygotes, this mutation also produces lengthened periods and arrhythmicity at the single-cell level.
Clock -/- null mutant mice, in which Clock has been knocked out, display completely normal circadian rhythms. The discovery of a null Clock mutant with a wild-type phenotype directly challenged the widely accepted premise that Clock is necessary for normal circadian function. Furthermore, it suggested that the CLOCK-BMAL1 dimer need not exist to modulate other elements of the circadian pathway. Neuronal PAS domain
PAS domain
The PAS domain is a protein domain contained in many signaling proteins where it functions as a signal sensor. PAS domains are found in a large number of organisms from bacteria to humans...
containing protein 2 (NPAS2
NPAS2
Neuronal PAS domain-containing protein 2 is a protein that in humans is encoded by the NPAS2 gene.-Interactions:NPAS2 has been shown to interact with Retinoic acid receptor alpha, EP300, ARNTL and Retinoid X receptor alpha....
, a CLOCK analog
Analogy (biology)
An analogy is a trait or an organ that appears similar in two unrelated organisms. The cladistic term for the same phenomenon is homoplasy, from Greek for same form. Biological anologies are often the result of convergent evolution....
) can substitute for CLOCK in these Clock-null mice.
Clock’s role in other feedback loops
The CLOCK-BMAL dimer is involved in regulation of other genes and feedback loops. An enzyme SIRT1 has also binds to the CLOCK-BMAL complex and acts to suppress its activity, perhaps by deacetylation of Bmal1 and surrounding histones. However, SIRT1’s role is still controversial and it may also have a role in deacetylating PER protein, targeting it for degradation.The CLOCK-BMAL dimer acts as a positive limb of a feedback loop. The binding of CLOCK-BMAL to an E-box promoter element activates transcription of clock genes such as per1, 2, and 3 and tim in mice. It has been shown in mice that CLOCK-BMAL also activates the Nicotinamide phosphoribosyltransferase
Nicotinamide phosphoribosyltransferase
Nicotinamide phosphoribosyltransferase also known as pre-B-cell colony-enhancing factor 1 or visfatin is an enzyme that in humans is encoded by the PBEF1 gene...
gene (also called Nampt), part of a separate feedback loop. This feedback loops creates a metabolic oscillator. The CLOCK-BMAL dimer activates transcription of the Nampt gene, which codes for the NAMPT protein. NAMPT is part of a series of enzymatic reactions that covert niacin
Niacin
"Niacin" redirects here. For the neo-fusion band, see Niacin .Niacin is an organic compound with the formula and, depending on the definition used, one of the forty to eighty essential human nutrients.Niacin is one of five vitamins associated with a pandemic deficiency disease: niacin deficiency...
(also called nicotinamide
Nicotinamide
Nicotinamide, also known as niacinamide and nicotinic acid amide, is the amide of nicotinic acid . Nicotinamide is a water-soluble vitamin and is part of the vitamin B group...
) to NAD
NAD
NAD may refer to:* No abnormality detected, a medical status description* No apparent distress, a status description in childbirth* NAD Electronics, a Canadian audio equipment manufacturer...
. SIRT1, which requires NAD for its enzymatic activity, then uses increased NAD levels to deacetylate BMAL1, suppressing it. This suppression results in less transcription of the NAMPT, less NAMPT protein, less NAD made, and therefore less SIRT1 and less suppression of the CLOCK-BMAL dimer. This dimer can again positively activate the Nampt gene transcription and the cycle continues, creating another oscillatory loop involving CLOCK-BMAL as positive elements. The key role that Clock plays in metabolic and circadian loops highlights the close relationship between metabolism and circadian clocks.
Other functions of Clock
In humans, a polymorphismPolymorphism (biology)
Polymorphism in biology occurs when two or more clearly different phenotypes exist in the same population of a species — in other words, the occurrence of more than one form or morph...
in Clock, rs6832769, may be related to the personality trait agreeableness
Agreeableness
Agreeableness is a tendency to be pleasant and accommodating in social situations. In contemporary personality psychology, agreeableness is one of the five major dimensions of personality structure, reflecting individual differences in concern for cooperation and social harmony. People who score...
. Another single nucleotide polymorphism
Single nucleotide polymorphism
A single-nucleotide polymorphism is a DNA sequence variation occurring when a single nucleotide — A, T, C or G — in the genome differs between members of a biological species or paired chromosomes in an individual...
(SNP) in Clock, 3111C, has been associated with diurnal preference. This SNP is also associated with increased insomnia
Insomnia
Insomnia is most often defined by an individual's report of sleeping difficulties. While the term is sometimes used in sleep literature to describe a disorder demonstrated by polysomnographic evidence of disturbed sleep, insomnia is often defined as a positive response to either of two questions:...
, difficulty losing weight, and recurrence of major depressive episodes in patients with bipolar disorder
Bipolar disorder
Bipolar disorder or bipolar affective disorder, historically known as manic–depressive disorder, is a psychiatric diagnosis that describes a category of mood disorders defined by the presence of one or more episodes of abnormally elevated energy levels, cognition, and mood with or without one or...
.
In mice, Clock has been implicated in sleep disorders, metabolism
Metabolism
Metabolism is the set of chemical reactions that happen in the cells of living organisms to sustain life. These processes allow organisms to grow and reproduce, maintain their structures, and respond to their environments. Metabolism is usually divided into two categories...
, pregnancy
Pregnancy
Pregnancy refers to the fertilization and development of one or more offspring, known as a fetus or embryo, in a woman's uterus. In a pregnancy, there can be multiple gestations, as in the case of twins or triplets...
, and mood disorders. Clock mutant mice sleep less than normal mice each day. The mice also display altered levels of plasma glucose
Glucose
Glucose is a simple sugar and an important carbohydrate in biology. Cells use it as the primary source of energy and a metabolic intermediate...
and rhythms in food intake. These mutants develop metabolic syndrome
Metabolic syndrome
Metabolic syndrome is a combination of medical disorders that, when occurring together, increase the risk of developing cardiovascular disease and diabetes. It affects one in five people in the United States and prevalence increases with age...
symptoms over time. Furthermore, Clock mutants demonstrate disrupted estrous cycle
Estrous cycle
The estrous cycle comprises the recurring physiologic changes that are induced by reproductive hormones in most mammalian placental females. Estrous cycles start after puberty in sexually mature females and are interrupted by anestrous phases or pregnancies...
s and increased rates of full-term pregnancy failure. Mutant Clock has also been linked to bipolar disorder
Bipolar disorder
Bipolar disorder or bipolar affective disorder, historically known as manic–depressive disorder, is a psychiatric diagnosis that describes a category of mood disorders defined by the presence of one or more episodes of abnormally elevated energy levels, cognition, and mood with or without one or...
-like symptoms in mice, including mania
Mania
Mania, the presence of which is a criterion for certain psychiatric diagnoses, is a state of abnormally elevated or irritable mood, arousal, and/ or energy levels. In a sense, it is the opposite of depression...
and euphoria
Euphoria
Euphoria is an emotional and mental state defined as a sense of great elation and well being.Euphoria may also refer to:* Euphoria , a genus of scarab beetles* Euphoria, a genus name previously used for the longan and other trees...
. Clock mutant mice also exhibit increased excitability of dopamine
Dopamine
Dopamine is a catecholamine neurotransmitter present in a wide variety of animals, including both vertebrates and invertebrates. In the brain, this substituted phenethylamine functions as a neurotransmitter, activating the five known types of dopamine receptors—D1, D2, D3, D4, and D5—and their...
neurons in reward centers of the brain. These results have led Dr. Colleen McClung to propose using Clock mutant mice as a model for human mood and behavior disorders.
The CLOCK-BMAL dimer has also been shown to activate reverse-erb receptor alpha (Rev-ErbA alpha
Rev-ErbA alpha
Rev-ErbA alpha also known as NR1D1 , is a protein that in humans is encoded by the NR1D1 gene.Rev-erbα is member of the Rev-ErbA family of nuclear receptors and is a transcriptional repressor...
) and retinoic acid orphan receptor alpha (ROR-alpha). REV-ERBα and RORα regulate Bmal by binding to retinoic acid-related orphan receptor response elements (ROREs) in its promoter.
In 2010, a Yale University team led by Dr. Yong Zhu found that variations in the epigenetics
Epigenetics
In biology, and specifically genetics, epigenetics is the study of heritable changes in gene expression or cellular phenotype caused by mechanisms other than changes in the underlying DNA sequence – hence the name epi- -genetics...
of the Clock gene may lead to an increased risk of breast cancer. It was found that in women with breast cancer, there was significantly less methylation of the Clock promoter region. It was also noted that this effect was greater in women with estrogen and progesterone receptor-negative tumors.
The CLOCK gene may also be a target for somatic mutations in microsatellite unstable colorectal cancers. In a study done in 2010 by researchers in the University of Helsinki, 53% of putative novel microsatellite instability target genes responsible for colorectal cancer contained CLOCK mutations.
See also
- BMALBMALBmal is a gene that encodes proteins regulating circadian rhythm. BMAL proteins form part of a basic-helix-loop-helix transcription factor. BMAL-1 dimerizes with CLOCK in vivo and transactivates gene expression of Period and Timeless in drosophila by binding to E-box elements in their promoters...
- Period genePeriod (gene)Period is a gene located on the X chromosome of Drosophila melanogaster. Oscillations in levels of both per transcript and its corresponding protein PER have a period of approximately 24 hours and together play a central role in the molecular mechanism of the Drosophila biological clock driving...
- Suprachiasmatic nucleusSuprachiasmatic nucleusThe suprachiasmatic nucleus or nuclei, abbreviated SCN, is a tiny region on the brain's midline, situated directly above the optic chiasm. It is responsible for controlling circadian rhythms...
- Timeless geneTimeless (gene)Timeless is a gene in Drosophila which encodes TIM, an essential protein that regulates circadian rhythms. Timeless mRNA and protein oscillate rhythmically with time as part of a transcription-translation [negative feedback] loop involving the period gene and its protein.-Discovery:Timeless was...
- PdfPigment dispersing factorPigment dispersing factor is a gene that encodes for the protein PDF, which is part of a large family of neuropeptides. The analogous hormone, pigment dispersing hormone was named for the diurnal pigment movement effect it has in crustacean retinal cells, and was initially discovered in the...