CLOCK

4H10957512753ENSG00000134852ENSMUSG00000029238O15516O08785NM_001267843NM_004898NM_007715NM_001289826NM_001305222NP_001254772NP_004889NP_001276755NP_001292151NP_031741Clock (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. 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. Research shows that the CLOCK gene plays a major role as an activator of downstream elements in the pathway critical to the generation 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. 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. 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 loop 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.