Molecular Biology of the Pacemaker

Until the 1990s, very little was known regarding the intracellular mechanisms in the pacemaker responsible for the generation of circadian rhythms. The first indication that the circadian pacemaker had a genetic underpinning occurred during a selection experiment by Drs. Konopka and Benzer. In their classic experiment, the frequency of pupal hatching in Drosophila was studied, leading to the identification of the first single-gene circadian clock mutant, the Period (Per) gene. To date, three mammalian orthologs, homologs of the fly Period gene, have been identified (mPer1, mPer2, and mPer3). Shortly after the discovery of the Period gene, several other mutants exhibiting altered or eliminated circadian rhythm were discovered, including timeless (tim) in Drosophila and frequency (frq) in Neurospora. The first mammalian clock gene, Clock, was discovered by Joseph Takahashi using a phenotype-driven N-ethyl-N-nitrosourea (ENU) mu-tagenesis screen. The Clock gene was identified by positional cloning followed by a functional transgenic BAC rescue, which served to confirm that the Clock gene was in fact responsible for the mutant phenotype. More recently, two additional mammalian Clock genes, the Cryptochrome genes (mCry1 and mCry2), have been described.

The Clock gene regulates two properties of the circadian clock: the endogenous circadian period and the persistence of rhythmicity. The protein encoded by the Clock gene, CLOCK, is a basic helix-loop-helix (bHLH) PER-ARNT-SIM (PAS) protein that belongs to a family of transcription factors. Shortly after the cloning of Clock, a CLOCK-interacting protein, BMAL, was identified using a yeast two-hybrid system. The CLOCK-BMAL heterodimer acts in a trans-activating fashion on E-box elements, which contain bHLH DNA-binding domains. Notably, both Period and the Cryptochromes contain an E-box sequence in the promoter region. By using a luciferase gene reporter assay, it was shown that the CLOCK-BMAL1 heterodimer, via transactivation of the E-box enhancer, drives the positive component of Per and Cry transcriptional oscillations. In addition, the CLOCK-BMAL1 heterodimer is thought to activate transcription of arginine vasopressin, a primary output marker for the circadian clock. It is not currently known what other Clock output genes are controlled by CLOCK-BMAL1; however, it is likely there are many. Both PER and CRY proteins block the ability of the CLOCK-BMAL1 dimer to activate Cry and Per promoters via the E-box, and, thus, a simple negative feedback loop begins to emerge. The rhythmic expression of PER proteins is thought to constitute the output of the oscillator, leading to the expression of Clock-controlled genes (Fig. 4). More recently, it was shown that, in mice, the two Cryptochrome genes were

Figure 4 An illustration of the contemporary model for the autoregulatory transcriptional-translational feedback loop thought to constitute the endogenous time-keeping mechanism. The relationship between variables is described in the text. [Modified from Lowrey, P. L., Shimomura, K., Antoch, M. P., Yamazaki, S., Zemenides, P. D., Ralph, M. R., Menaker, M., and Takahashi, J. S. (2000). Positional syntenic cloning and functional characterization of the mammalian circadian mutation tau. Science 288, 483-492.]

Figure 4 An illustration of the contemporary model for the autoregulatory transcriptional-translational feedback loop thought to constitute the endogenous time-keeping mechanism. The relationship between variables is described in the text. [Modified from Lowrey, P. L., Shimomura, K., Antoch, M. P., Yamazaki, S., Zemenides, P. D., Ralph, M. R., Menaker, M., and Takahashi, J. S. (2000). Positional syntenic cloning and functional characterization of the mammalian circadian mutation tau. Science 288, 483-492.]

essential components of the negative limb of the circadian clock feedback loop. It now appears that the Cryptochrome proteins, CRY1 and CRY2, play an important role in the translocation of PER back to the nucleus, where PER disrupts CLOCK-BMAL1 tran-scriptional activity. A mammalian circadian mutant, tau, has been described. The tau mutation is a semidominant autosomal allele, which shortens the period length of circadian rhythms in Syrian hamsters. It was determined that the tau locus encodes casein kinase I epsilon (CKIe). CKIe phosphorylates PER proteins, which targets them for degradation. CKIe is the only enzyme identified so far in the mammalian pacemaker and, thus, may be a potential target for pharmaceutical compounds. Interestingly, the tim gene does not appear to have a role in mammalian circadian time-keeping.

How do light during the night and behavioral events during the day produce delays and advances? Evidence suggests that both types of shifts are mediated by changes in the levels of the state variables of the clock. For example, it now appears that PER1 protein and mRNA are rapidly down-regulated during behavioral resetting and that PER1 protein and mRNA are rapidly induced after light exposure. Whereas it is clear that much remains to be discovered about the mechanisms by which the clock loop is modulated, it is known that light appears to differentially regulate the Per gene. Future studies utilizing conditional transgenic and knockout mice in which genes are altered in a development- or tissue-specific manner should prove to be powerful tools for the dissection of the mammalian circadian clock mechanisms.

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