Clock (Circadian Locomotor Output Cycles Kaput) is a gene encoding a basic helix-loop-helix-PAS transcription factor (CLOCK) that is believed to affect both the persistence and period of circadian rhythms.
The Clock gene was first identified in 1994 by Dr. Joseph Takahashi 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. Mice bred to be heterozygous showed longer periods of 24.4 hours compared to the control 23.3 hour period. Mice homozygous for the mutation showed 27.3 hour periods, but eventually lost all circadian rhythmicity after several days in constant darkness. That showed that "intact Clock genes" are necessary for normal mammalian circadian function[how?].
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 nuclear 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 domains. 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 doubletime (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. 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 chrystallographic analysis.
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.
Role in other feedback loops
The CLOCK-BMAL dimer is involved in regulation of other genes and feedback loops. An enzyme SIRT1 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 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 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 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.
Clock mutant organisms can either possess a null mutation or an antimorphic 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.
In Drosophila, a mutant form of Clock (Jrk) was identified by Allada, Hall, and Rosbash in 1998. The team used forward genetics to identify non-circadian rhythms in mutant flies. Jrk results from a premature stop 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 that Clock 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. The similar sequence between Jrk and its mouse homolog suggests common circadian rhythm components were present in both Drosophila and mice ancestors. A recessive allele of Clock leads to behavioral arrhythmicity while maintaining detectable molecular and transcriptional oscillations. This suggests that Clk contributes to the amplitude of circadian rhythms.
The mouse homolog 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 paralog) can substitute for CLOCK in these Clock-null mice. Mice with one NPAS2 allele showed shorter periods at first, but eventual arrhythmic behavior.
In humans, a polymorphism 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 cycles 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.
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. Approximately half of putative novel microsatellite instability target genes responsible for colorectal cancer contained CLOCK mutations. 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, with quantitative physical responses to circadian food intake as potential inputs to the clock system.
- Dunlap JC (Jan 1999). "Molecular bases for circadian clocks". Cell. 96 (2): 271–90. doi:10.1016/S0092-8674(00)80566-8. PMID 9988221.
- King DP, Zhao Y, Sangoram AM, Wilsbacher LD, Tanaka M, Antoch MP, Steeves TD, Vitaterna MH, Kornhauser JM, Lowrey PL, Turek FW, Takahashi JS (May 1997). "Positional cloning of the mouse circadian clock gene". Cell. 89 (4): 641–653. doi:10.1016/S0092-8674(00)80245-7. PMC 3815553. PMID 9160755.
- Vitaterna MH, King DP, Chang AM, Kornhauser JM, Lowrey PL, McDonald JD, Dove WF, Pinto LH, Turek FW, Takahashi JS (Apr 1994). "Mutagenesis and mapping of a mouse gene, Clock, essential for circadian behavior". Science. 264 (5159): 719–25. doi:10.1126/science.8171325. PMC 3839659. PMID 8171325.
- Hardin PE (2000). "From biological clock to biological rhythms". Genome Biology. 1 (4): 1023.1–1023.5. doi:10.1186/gb-2000-1-4-reviews1023. PMC 138871. PMID 11178250.
- Hung HC, Maurer C, Zorn D, Chang WL, Weber F (Aug 2009). "Sequential and compartment-specific phosphorylation controls the life cycle of the circadian CLOCK protein". The Journal of Biological Chemistry. 284 (35): 23734–42. doi:10.1074/jbc.M109.025064. PMC 2749147. PMID 19564332.
- Yu W, Zheng H, Houl JH, Dauwalder B, Hardin PE (Mar 2006). "PER-dependent rhythms in CLK phosphorylation and E-box binding regulate circadian transcription". Genes & Development. 20 (6): 723–33. doi:10.1101/gad.1404406. PMC 1434787. PMID 16543224.
- Gekakis N, Staknis D, Nguyen HB, Davis FC, Wilsbacher LD, King DP, Takahashi JS, Weitz CJ (Jun 1998). "Role of the CLOCK protein in the mammalian circadian mechanism". Science. 280 (5369): 1564–9. doi:10.1126/science.280.5369.1564. PMID 9616112.
- Huang N, Chelliah Y, Shan Y, Taylor CA, Yoo SH, Partch C, Green CB, Zhang H, Takahashi JS (Jul 2012). "Crystal structure of the heterodimeric CLOCK:BMAL1 transcriptional activator complex". Science. 337 (6091): 189–94. doi:10.1126/science.1222804. PMC 3694778. PMID 22653727.
- Doi M, Hirayama J, Sassone-Corsi P (May 2006). "Circadian regulator CLOCK is a histone acetyltransferase". Cell. 125 (3): 497–508. doi:10.1016/j.cell.2006.03.033. PMID 16678094.
- Nakahata Y, Kaluzova M, Grimaldi B, Sahar S, Hirayama J, Chen D, Guarente LP, Sassone-Corsi P (Jul 2008). "The NAD+-dependent deacetylase SIRT1 modulates CLOCK-mediated chromatin remodeling and circadian control". Cell. 134 (2): 329–40. doi:10.1016/j.cell.2008.07.002. PMC 3526943. PMID 18662547.
- Asher G, Gatfield D, Stratmann M, Reinke H, Dibner C, Kreppel F, Mostoslavsky R, Alt FW, Schibler U (Jul 2008). "SIRT1 regulates circadian clock gene expression through PER2 deacetylation". Cell. 134 (2): 317–28. doi:10.1016/j.cell.2008.06.050. PMID 18662546.
- Ramsey KM, Yoshino J, Brace CS, Abrassart D, Kobayashi Y, Marcheva B, Hong HK, Chong JL, Buhr ED, Lee C, Takahashi JS, Imai S, Bass J (May 2009). "Circadian clock feedback cycle through NAMPT-mediated NAD+ biosynthesis". Science. 324 (5927): 651–4. doi:10.1126/science.1171641. PMC 2738420. PMID 19299583.
- Panda S, Antoch MP, Miller BH, Su AI, Schook AB, Straume M, Schultz PG, Kay SA, Takahashi JS, Hogenesch JB (May 2002). "Coordinated transcription of key pathways in the mouse by the circadian clock". Cell. 109 (3): 307–20. doi:10.1016/S0092-8674(02)00722-5. PMID 12015981.
- Allada R, White NE, So WV, Hall JC, Rosbash M (May 1998). "A mutant Drosophila homolog of mammalian Clock disrupts circadian rhythms and transcription of period and timeless". Cell. 93 (5): 791–804. doi:10.1016/S0092-8674(00)81440-3. PMID 9630223.
- Kraupp VO (Jan 1975). "[Pharmacodynamic examples on the effect enhancement or alteration of action through molecular dimerization]". Wiener Medizinische Wochenschrift. 125 (1–3): 3367–3375. doi:10.1093/emboj/cdg318. PMC 165643. PMID 165643.
- Herzog ED, Takahashi JS, Block GD (Dec 1998). "Clock controls circadian period in isolated suprachiasmatic nucleus neurons". Nature Neuroscience. 1 (8): 708–13. doi:10.1038/3708. PMID 10196587.
- Debruyne JP, Noton E, Lambert CM, Maywood ES, Weaver DR, Reppert SM (May 2006). "A clock shock: mouse CLOCK is not required for circadian oscillator function". Neuron. 50 (3): 465–77. doi:10.1016/j.neuron.2006.03.041. PMID 16675400.
- Debruyne JP (Dec 2008). "Oscillating perceptions: the ups and downs of the CLOCK protein in the mouse circadian system". Journal of Genetics. 87 (5): 437–446. doi:10.1007/s12041-008-0066-7. PMC 2749070. PMID 19147932.
- DeBruyne JP, Weaver DR, Reppert SM (May 2007). "CLOCK and NPAS2 have overlapping roles in the suprachiasmatic circadian clock". Nature Neuroscience. 10 (5): 543–5. doi:10.1038/nn1884. PMC 2782643. PMID 17417633.
- Terracciano A, Sanna S, Uda M, Deiana B, Usala G, Busonero F, Maschio A, Scally M, Patriciu N, Chen WM, Distel MA, Slagboom EP, Boomsma DI, Villafuerte S, Sliwerska E, Burmeister M, Amin N, Janssens AC, van Duijn CM, Schlessinger D, Abecasis GR, Costa PT (Jun 2010). "Genome-wide association scan for five major dimensions of personality". Molecular Psychiatry. 15 (6): 647–56. doi:10.1038/mp.2008.113. PMC 2874623. PMID 18957941.
- Katzenberg D, Young T, Finn L, Lin L, King DP, Takahashi JS, Mignot E (Sep 1998). "A CLOCK polymorphism associated with human diurnal preference". Sleep. 21 (6): 569–76. PMID 9779516.
- Serretti A, Benedetti F, Mandelli L, Lorenzi C, Pirovano A, Colombo C, Smeraldi E (Aug 2003). "Genetic dissection of psychopathological symptoms: insomnia in mood disorders and CLOCK gene polymorphism". American Journal of Medical Genetics Part B. 121B (1): 35–8. doi:10.1002/ajmg.b.20053. PMID 12898572.
- Garaulet M, Corbalán MD, Madrid JA, Morales E, Baraza JC, Lee YC, Ordovas JM (Mar 2010). "CLOCK gene is implicated in weight reduction in obese patients participating in a dietary programme based on the Mediterranean diet". International Journal of Obesity. 34 (3): 516–23. doi:10.1038/ijo.2009.255. PMC 4426985. PMID 20065968.
- Bunney BG, Li JZ, Walsh DM, Stein R, Vawter MP, Cartagena P, Barchas JD, Schatzberg AF, Myers RM, Watson SJ, Akil H, Bunney WE (Feb 2015). "Circadian dysregulation of clock genes: clues to rapid treatments in major depressive disorder". Molecular Psychiatry. 20 (1): 48–55. doi:10.1038/mp.2014.138. PMC 4765913. PMID 25349171.
- Naylor E, Bergmann BM, Krauski K, Zee PC, Takahashi JS, Vitaterna MH, Turek FW (Nov 2000). "The circadian clock mutation alters sleep homeostasis in the mouse". The Journal of Neuroscience. 20 (21): 8138–43. PMID 11050136.
- Turek FW, Joshu C, Kohsaka A, Lin E, Ivanova G, McDearmon E, Laposky A, Losee-Olson S, Easton A, Jensen DR, Eckel RH, Takahashi JS, Bass J (May 2005). "Obesity and metabolic syndrome in circadian Clock mutant mice". Science. 308 (5724): 1043–1045. doi:10.1126/science.1108750. PMC 3764501. PMID 15845877.
- Turek FW, Joshu C, Kohsaka A, Lin E, Ivanova G, McDearmon E, Laposky A, Losee-Olson S, Easton A, Jensen DR, Eckel RH, Takahashi JS, Bass J (May 2005). "Obesity and metabolic syndrome in circadian Clock mutant mice". Science. 308 (5724): 1043–5. doi:10.1126/science.1108750. PMC 3764501. PMID 15845877.
- Miller BH, Olson SL, Turek FW, Levine JE, Horton TH, Takahashi JS (Aug 2004). "Circadian clock mutation disrupts estrous cyclicity and maintenance of pregnancy". Current Biology. 14 (15): 1367–73. doi:10.1016/j.cub.2004.07.055. PMC 3756147. PMID 15296754.
- McClung CA (May 2007). "Circadian genes, rhythms and the biology of mood disorders". Pharmacology & Therapeutics. 114 (2): 222–32. doi:10.1016/j.pharmthera.2007.02.003. PMC 1925042. PMID 17395264.
- McClung CA, Sidiropoulou K, Vitaterna M, Takahashi JS, White FJ, Cooper DC, Nestler EJ (Jun 2005). "Regulation of dopaminergic transmission and cocaine reward by the Clock gene". Proceedings of the National Academy of Sciences of the United States of America. 102 (26): 9377–81. doi:10.1073/pnas.0503584102. PMC 1166621. PMID 15967985.
- Preitner N, Damiola F, Lopez-Molina L, Zakany J, Duboule D, Albrecht U, Schibler U (Jul 2002). "The orphan nuclear receptor REV-ERBalpha controls circadian transcription within the positive limb of the mammalian circadian oscillator". Cell. 110 (2): 251–60. doi:10.1016/S0092-8674(02)00825-5. PMID 12150932.
- Guillaumond F, Dardente H, Giguère V, Cermakian N (Oct 2005). "Differential control of Bmal1 circadian transcription by REV-ERB and ROR nuclear receptors". Journal of Biological Rhythms. 20 (5): 391–403. doi:10.1177/0748730405277232. PMID 16267379.
- Dodson, Helen. "Women With Variants in "CLOCK" Gene Have Higher Risk of Breast Cancer". Yale Office of Public Affairs and Communications. Archived from the original on 2011-07-24. Retrieved 21 April 2011.
- Joska TM, Zaman R, Belden WJ (September 2014). "Regulated DNA methylation and the circadian clock: implications in cancer". Biology. 3 (3): 560–577. doi:10.3390/biology3030560. PMC 4192628. PMID 25198253.
- Alhopuro P, Björklund M, Sammalkorpi H, Turunen M, Tuupanen S, Biström M, Niittymäki I, Lehtonen HJ, Kivioja T, Launonen V, Saharinen J, Nousiainen K, Hautaniemi S, Nuorva K, Mecklin JP, Järvinen H, Orntoft T, Arango D, Lehtonen R, Karhu A, Taipale J, Aaltonen LA (Jul 2010). "Mutations in the circadian gene CLOCK in colorectal cancer". Molecular Cancer Research. 8 (7): 952–60. doi:10.1158/1541-7786.MCR-10-0086. PMID 20551151.
- Yoshino J, Klein S (Jul 2013). "A Novel Link Between Circadian Clocks and Adipose Tissue Energy Metabolism". Diabetes. 62 (7): 2175–7. doi:10.2337/db13-0457. PMC 3712037. PMID 23801717.
- Johnston J (Jun 2014). "Physiological responses to food intake throughout the day". Nutrition Research Reviews. 27 (1): 107–118. doi:10.1017/S0954422414000055. PMC 4078443. PMID 24666537.