CLOCK (backronym forcircadian locomotor output cycles kaput) is agene encoding abasic helix-loop-helix-PAStranscription factor that is known to affect both the persistence andperiod ofcircadian rhythms.
Research shows that theCLOCK gene plays a major role as anactivator of downstream elements in the pathway critical to the generation ofcircadian rhythms.[5][6]
TheCLOCK gene was first identified in 1997 byJoseph Takahashi and his colleagues. Takahashi used forward mutagenesis screening of mice treated withN-ethyl-N-nitrosourea to create and identifymutations in key genes that broadly affect circadian activity.[7] TheCLOCK mutants discovered through the screen displayed an abnormally long period of daily activity. This trait proved to beheritable. Mice bred to beheterozygous showed longer periods of 24.4 hours compared to the control 23.3 hour period. Micehomozygous for the mutation showed 27.3 hour periods, but eventually lost all circadian rhythmicity after several days in constant darkness.[8] That showed that "intactCLOCK genes" are necessary for normal mammalian circadian function, as these mutations were semidominant.[8]
CLOCK protein has been found to play a central role as a transcription factor in the circadian pacemaker.[9] InDrosophila, newly synthesized CLOCK (CLK) is hypophosphorylated in thecytoplasm before entering the nucleus. Once in the nuclei, CLK is localized in nuclear foci and is later redistributed homogeneously. CYCLE (CYC) (also known as dBMAL for theBMAL1ortholog in mammals) dimerizes with CLK via their respectivePAS domains. This dimer then recruits co-activatorCREB-binding protein (CBP) and is further phosphorylated.[10] Once phosphorylated, this CLK-CYC complex binds to theE-box elements of the promoters ofperiod (per) andtimeless (tim) via its bHLH domain, causing the stimulation of gene expression ofper andtim. 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 ofper andtim, essentially blockingper andtim transcription.[6][11] CLK ishyperphosphorylated whendoubletime (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.[11] Hypophosphorylated CLK then accumulates, binds to the E-boxes ofper andtim and activates their transcription once again.[11] This cycle of post-translational phosphorylation suggest that temporal phosphorylation of CLK helps in the timing mechanism of the circadian clock.[10]
A similar model is found in mice, in which BMAL1 dimerizes with CLOCK to activateper andcryptochrome (cry) transcription. PER and CRY proteins form a heterodimer which acts on the CLOCK-BMAL heterodimer to repress the transcription ofper andcry.[12] The heterodimer CLOCK:BMAL1 functions similarly to other transcriptional activator complexes; CLOCK:BMAL1 interacts with the E-box regulatory elements. PER and CRY proteins accumulate and dimerize during subjective night, and translocate into the nucleus to interact with the CLOCK:BMAL1 complex, directly inhibiting their own expression. This research has been conducted and validated through crystallographic analysis.[13]
CLOCK exhibitshistone acetyl transferase (HAT) activity, which is enhanced bydimerization with BMAL1.[14] Dr. Paolo Sassone-Corsi and colleagues demonstratedin vitro that CLOCK mediated HAT activity is necessary to rescue circadian rhythms in Clock mutants.[14]
The CLOCK-BMAL dimer is involved in regulation of other genes and feedback loops. An enzymeSIRT1 also binds to the CLOCK-BMAL complex and acts to suppress its activity, perhaps bydeacetylation ofBmal1 and surroundinghistones.[15] However, SIRT1's role is still controversial and it may also have a role in deacetylating PER protein, targeting it for degradation.[16]
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 asper1, 2, and 3 andtim in mice. It has been shown in mice that CLOCK-BMAL also activates theNicotinamide phosphoribosyltransferase gene (also calledNampt), part of a separate feedback loop. This feedback loop creates a metabolic oscillator. The CLOCK-BMAL dimer activates transcription of theNampt gene, which codes for the NAMPT protein. NAMPT is part of a series of enzymatic reactions that covertniacin (also callednicotinamide) toNAD. SIRT1, which requires NAD for its enzymatic activity, then uses increased NAD levels to suppress BMAL1 through deacetylation. 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 theNampt gene transcription and the cycle continues, creating another oscillatory loop involving CLOCK-BMAL as positive elements. The key role thatClock plays in metabolic and circadian loops highlights the close relationship between metabolism and circadian clocks.[17]
The firstcircadian rhythms were most likely generated by light-driven cell division cycles in ancestralprokaryotic species.[18] This proto-rhythm later evolved into a self-sustaining clock viagene duplication and functional divergence of clock genes. ThekaiA/B/C gene clusters remain the oldest of the clock genes as they are present incyanobacteria, withkaiC most likely the ancestor ofkaiA andkaiB.[18] The function of these ancestral clock genes was most likely related to chromosome function before evolving a timing mechanism.[18] ThekaiA andkaiB genes arose after cyanobacteria separated from other prokaryotes.[19] Harsh climate conditions in the early history of the Earth's formation, such as UV irradiation, may have led to the diversification of clock genes in prokaryotes in response to drastic changes in climate.[19]
Cryptochromes, light-sensitive proteins regulated byCry genes, are most likely descendents ofkaiC resulting from a genome duplication predating theCambrian explosion and are responsible for negative regulation of circadian clocks. Other distinct clock gene lineages arose early in vertebrate evolution, with gene BMAL1paralogous to CLOCK. Their common ancestor, however, most likely predated theinsect-vertebrate split roughly 500 mya.[18] WC1, an analog of CLOCK/BMAL1 found in fungal genomes, is a proposed candidate common ancestor predating thefungi-animal split.[18] A BLAST search conducted in a 2004 review of clock gene evolution suggested theClock gene may have arisen from a duplication in the BMAL1 gene, though this hypothesis remains speculative.[18] Another theory alternatively proposes the NPAS2 gene as the paralog of CLOCK that performs a similar role in the circadian rhythm pathway but in different tissues.[20]
Allelic variations within the Clock1a gene in particular are hypothesized to have effects on seasonal timing according to a 2014 study conducted in a population of cyprinid fishes.[21] Polymorphisms in the gene mainly affect the length of the PolyQ domain region, providing an example ofdivergent evolution where species sharing anecological niche will partition resources in seasonally variable environments.[21] The length of the PolyQ domain is associated with changes in transcription level of CLOCK. On average, longer allele lengths were correlated with recently derived species and earlier-spawning species, most likely due to seasonal changes in water temperature.[21] The researchers hypothesize that the length of the domain may serve to compensate for changes in temperature by altering the rate of CLOCK transcription. All other amino acids remained identical across native species, indicating that functional constraint may be another factor influencing CLOCK gene evolution in addition to gene duplication anddiversification.[20][21]
One 2017 study investigating the role of CLOCK expression in neurons determined its function in regulating transcriptional networks that could provide insight into human brain evolution.[22] The researchers synthesized differentiated human neuronsin vitro and then performedgene knockdown to test the effect of CLOCK on neuronal cell signaling. When CLOCK activity was disrupted, increased neuronal migration of tissue in theneocortex was observed, suggesting a molecular mechanism forcortical expansion unique to human brain development.[22] However, the precise role of CLOCK in metabolic regulation of cortical neurons remains to be determined. Another study looking at the relationship between CLOCK polymorphisms in the3' flanking region and morning/evening preference in adults found a correlation between subjects with the 3111C allele and preference for evening hours based on answers provided in a scored questionnaire.[23] This region is well conserved between mice and humans andpolymorphisms have been shown to affect mRNA stability, indicating allelic variants could disrupt normal circadian patterns in mammals leading to conditions such asinsomnia or other sleep disorders.[23]
Clock mutant organisms can either possess a null mutation or anantimorphic allele at theClock locus that codes for an antagonist to the wild-type protein. The presence of an antimorphic protein downregulates the transcriptional products normally upregulated byClock.[24]
InDrosophila, a mutant form ofClock (Jrk) was identified by Allada,Hall, andRosbash in 1998. The team usedforward genetics to identify non-circadian rhythms in mutant flies.Jrk results from a prematurestop codon that eliminates the activation domain of the CLOCK protein. This mutation causes dominant effects: half of the heterozygous flies with this mutant gene have a lengthened period of 24.8 hours, while the other half become arrhythmic. Homozygous flies lose their circadian rhythm. Furthermore, the same researchers demonstrated that these mutant flies express low levels of PER and TIM proteins, indicating thatClock functions as a positive element in the circadian loop. While the mutation affects the circadian clock of the fly, it does not cause any physiological or behavioral defects.[25] The similar sequence betweenJrk and its mousehomolog suggests common circadian rhythm components were present in bothDrosophila and mice ancestors. A recessive allele ofClock leads to behavioral arrhythmicity while maintaining detectable molecular and transcriptional oscillations. This suggests thatClk contributes to the amplitude of circadian rhythms.[26]
The mouse homolog to theJrk mutant is theClockΔ19 mutant that possesses a deletion in exon 19 of theClock gene. This dominant-negative mutation results in a defective CLOCK-BMAL dimer, which causes mice to have a decreased ability to activateper transcription. In constant darkness,ClockΔ19 mice heterozygous for theClock mutant allele exhibit lengthened circadian periods, whileClockΔ19/Δ19 mice homozygous for the allele become arrhythmic.[8] In both heterozygotes and homozygotes, this mutation also produces lengthened periods and arrhythmicity at the single-cell level.[27]
Clock -/- null mutant mice, in whichClock has been knocked out, display completely normal circadian rhythms. The discovery of a nullClock mutant with a wild-type phenotype directly challenged the widely accepted premise thatClock 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.[28] Neuronal PAS domain containing protein 2 (NPAS2, a CLOCKparalog[29]) can substitute for CLOCK in theseClock-null mice. Mice with one NPAS2allele showed shorter periods at first, but eventual arrhythmic behavior.[30]
In humans, apolymorphism inClock, rs6832769, may be related to thepersonality traitagreeableness.[31] Anothersingle nucleotide polymorphism (SNP) inClock, 3111C, associated withdiurnal preference,[23] is also associated with increasedinsomnia,[32] difficulty losing weight,[33] and recurrence of major depressive episodes in patients withbipolar disorder.[34]
In mice,Clock has been implicated insleep disorders,metabolism,pregnancy, andmood disorders.Clock mutant mice sleep less than normal mice each day.[35] The mice also display altered levels of plasmaglucose and rhythms in food intake.[36] These mutants developmetabolic syndrome symptoms over time.[36] Furthermore,Clock mutants demonstrate disruptedestrous cycles and increased rates of full-term pregnancy failure.[37] MutantClock has also been linked to bipolar disorder-like symptoms in mice, includingmania andeuphoria.[38]Clock mutant mice also exhibit increased excitability ofdopamine neurons in reward centers of the brain.[39] These results have ledColleen McClung to propose usingClock 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 regulateBmal by binding to retinoic acid-related orphan receptor response elements (ROREs) in its promoter.[40][41]
Variations in theepigenetics of theClock gene may lead to an increased risk ofbreast cancer.[42] It was found that in women with breast cancer, there was significantly less methylation of theClock promoter region. It was also noted that this effect was greater in women with estrogen and progesterone receptor-negative tumors.[43]
The CLOCK gene may also be a target for somatic mutations in microsatellite unstablecolorectal cancers. In one study, 53% of microsatellite instability colorectal cancer cases contained somatic CLOCK mutations.[44] Nascent research in the expression of circadian genes in adipose tissue suggests that suppression of the CLOCK gene may causally correlate not only with obesity, but also with type 2 diabetes,[45] with quantitative physical responses to circadian food intake as potential inputs to the clock system.[46]
