Clock Mechanism in Mammals Flashcards
What is the overall clock mechanism in mammals?
The cell-autonomous molecular clock in mammals is generated by two interlocking transcription/translation feedback loops (TTFL) that function together to produce robust 24-hour rhythms of gene expression. The core TTFL is driven by four integral clock proteins: two activators (CLOCK and BMAL1) and two repressors (PER and CRY), as well as kinases and phosphatases that regulate the localization and stability of these integral clock proteins.
CLOCK and BMAL1, which belong to the family of bHLH-PAS-containing transcription factors, bind and form a heterodimer. They attach to specific E boxes, which, if linked to a promoter, will cause transcription of downstream genes. The E-box sequence is quite regular, explaining why half of our genome has been found rhythmic.
CLOCK:BMAL activates transcription of the repressor Per and Cry genes. As PER and CRY proteins accumulate, they heterodimerize in the cytoplasm and translocate to the nucleus to interact with CLOCK:BMAL1, inhibiting further transcriptional activation.
The casein kinase 1 epsiolin can bind to and phosphorylate PER. Phosphorylation of PERs creates binding sites for an ubiquitin ligase resulting in its subsequent degradation. As PER and CRY proteins are degraded through ubiquitin-dependent pathways, repression on CLOCK:BMAL1 is relieved and the cycle begins again with ~24-hour periodicity.
As a result, the casein kinases CKIδ and CKIε play an important role in determining the intrinsic period of the clock by controlling the rate at which the PER:CRY complexes are either degraded or enter the nucleus.
What changes in the molecular clock can lead to changes in the observed period of an animal?
Posttranslational modifications are important in determining the period of a circadian rhythm.
Lowrey et al., (2000) showed that in the tau mutant hamster, due to a point mutation C->T, CK1e is unable to phosphorylate the PER proteins. Hypophopshorylated PER is translocated to the nucleus , where its accumulation leads to earlier repression of CLOCK:BMAL -mediated transcription, shorterning the circadian period to 20 hours.
Researchers noticed that not only PER but also CRY stability is important in determining circadian period. Siepka et al., (2007) found a circadian mutant, named Overtime, with a long period of 26 hours. Positional cloning and genetic complementation revealed that Overtime is encoded by the F-box protein FBXL3, a member of the (SCF) E3 ubiquitin ligases which bind to CRY and decrease its half life. The overtime mutant leads to a loss of function mutation in FBXL3, leading to reduced degradation and increased stability of CRY. This in turn leads to a global transcriptional repression of the Per and Cry genes and therefore longer repression of CLOCK-BMAL1- dependent gene transcription.
Loss of function mutations in CK1e will result in an increase in the rate of accumulation of PER, so will shorten the intrinsic period of the clock in mice and give rise to sleep phase disorders in humans
What did Etchegaray et al., 2003 show?
They showed that rhythmic histone (H3) acetylation underlies transcription in the mammalian circadian clock. It was found that the promoter regions of Per1, Per2 and Cry1 genes show circadian rhythms in H3 acetylation, which correlate with mRNA rhythms.
It was also found that the histone acetyltransferase p300 precipitates together with Clock in vivo in a time-dependent manner. Moreover, the Cry proteins inhibit a p300-induced increase in Clock/Bmal1-mediated transcription
How is the oscillatory expression of clock-controlled genes regulated, so that transcription-permissive chro- matin states are dynamically established in a circadian time-specific manner?
The activation of clock-controlled genes by CLOCK:BMAL1 has been shown to be coupled to circadian changes in histone acetylation at their promoters (Etchegaray et al. 2003). Specifically, histone H3 is acetylated in chromatin that encompasses the Per1, Per2, and Cry1 promoters when these genes are actively transcribed.
Thus, chromatin modelling is necessary for cyclic transcriptional activity.
Takahashi et al., 1993?
founf the first clock mutant mouse
King et al., 1997?
They used used positional cloning to identify the circadian Clock gene in mice. In the Clock mutant allele, an A→T single point mutation caused splicing out of exon 19 from the gene and deletion of 51 amino acids in the CLOCK protein. They found that this caused a number of circadian changes but most notably in homozygous mutants, there was a 3-4 hr lengthening of the peirod and a loss rhythmicity in constant darkness.
This group also identified specific regions within the gene which allowed them to predict how it contributes to circadiac function- e.g. regions found predicting DNA binding, protein dimerization, and activation domains.
Doi et al., 2006?
They showed that Clock has a histone acetyl transferase domain and that its HAT activity/function is essential for the circadian control of clock-controlled genes.
They used an experimental system based on mouse embryonic fibroblast (MEF) cells derived from homozygous Clock mutant (c/c) mice. MEF c/c cells show no cyclic expression of clock genes (Pando et al. 2002). However Dio et al., (2006) demonstrated that ectopic expression of CLOCK is able to rescue the circadian expression of endogenous target genes (Per1/ Ddp). In contrast, ectopic expression of a HAT-deficient CLOCK failed to restore the circadian transactivation of mPer1and Dbp, demonstrating that the HAT activity of CLOCK is necessary for circadian gene expression.
How was BMA1 discovered?
The presence of the PAS dimerization domain in CLOCK protein suggested that it may form a heterodimer. A screen for potential partners for the CLOCK protein using the yeast two-hybrid system revealed that Bmal1 is coexpressed with clock in the SCN and retina. In addition they found that BMAL1 was able to dimerize with the CLOCK protein and activate transcription when it binds with e-box elements (Gekakis et al., 1998).
Bunger et al., 2000
KO BMAL-1 mice demonstrated the critical role of this gene in circadian rhythm generation. These mutant mice, while being able to entrain to the light–dark cycle, became arrhythmic immediately upon release in constant darkness (Bunger et al., 2000).
These findings confirmed that BMAL1 is a central and nonredundant component of the molecular clock
Period gene?
The Period gene, which was first characterized in Drosophila (Reddy et al. 1984), is a core molecular component of the circadian system of animals. In vertebrates (mammals), the Period gene homologs—Per1, Per2, and Per3—have been identified, and their roles as circadian oscillators have been extensively studied (Zylka et al. 1998).
In mammals, Per1 and Per2 appear to be oscillatory genes that are indispensable for generation of the circadian rhythm. Homozygous mouse mutants of either mPer1 or mPer2 severely disrupted locomotor activities during extended exposure to constant dark conditions, while knockout of mPer3 caused subtle effects on such activities (Bae et al. 2000; Shearman et al. 2000).
After exposure to light during the subjective night, transcription of mPer1 and mPer2 was rapidly induced in the suprachiasmatic nucleus (SCN) (Zylka et al. 1998), suggesting that these genes are involved in the light resetting of the circadian oscillator.
Cry gene?
The mammalian proteins Cryl and Cry2, are members of the family of plant blue-light receptors (cryptochromes) and photolyases. van de Horst et al., (1999) proposed that they are the candidate light receptors for photoentrainment of the biological clock.
This is because they demonstarted that mice lacking the Cryl (cry1 KO) or Cry2 protein (Cry2 KO) display accelerated and delayed free-running periodicity of locomotor activity, respectively. This suggests that Cry1 and Cry2 have an antagonist clock-adjusting function.
Strikingly, in the absence of both proteins (double KO), an instantaneous and complete loss of free-running rhythmicity is observed. This suggests that, both proteins are essential for the maintenance of circadian rhythmicity (van de Horst et al., 1999)
