Molecular Basis Of Circadian Organization

There has been remarkable progress in the past 15 years in identifying the molecular basis of circadian clocks in a variety of organisms. In animals, much of this progress has resulted from pioneering work with the fruit fly. In 1971 the first clock gene, the period gene, was discovered in a mutagenesis screen in D. melanogaster. A decade later the gene was cloned, paving the way for studies of the gene's regulation. This work led to the discovery of several other genes in D. melanogaster that appear to be part of the clock mechanism, including those involved in entrainment.

Molecular Basis of the Clock

Four genes, the transcriptional regulators period (per), timeless (tim), cycle (cyc), and clock (clk), have been shown to be critical components for generating the basic circadian oscillation. Of the four, three, per, tim, and clk, are rhythmically expressed and circadian oscillations in both mRNA and protein levels are well documented. The fourth gene, cyc, is expressed at relatively constant levels throughout the day. Both CLK and CYC proteins are transcription factors that utilize basic helix-loop-helix domains to bind to E boxes, and both contain protein—protein interaction domains (PAS domains) that likely mediate the association of the two proteins with each other, thus forming heterodimers. The fundamental mechanism for generating the oscillation involves a transcription/ translation negative feedback loop. The basic loop is illustrated in Fig. 3. A heterodimer composed of CLK and CYC binds to promoters of per and tim, leading to an increase in transcription of these two genes that continues throughout the day. Levels of mRNA for the

FIGURE 3 Molecular model of the circadian pacemaker of Drosophila showing the proposed negative feedback loop of the oscillation. CLK and CYC heterodimers bind to E boxes of nuclear DNA promoting transcription of per and tim genes. TIM and PER proteins heterodimerize and are phosphorylated by DBT. The heterodimer enters the nucleus and inhibits the positive regulation by the CLK/CYC heterodimer. Light enters the system through CRY and promotes turnover of TIM and PER (modified from Dunlap, 1999).

two genes peak in the early night. Protein products of these two genes increase as well, but peak levels of protein are delayed by several hours, peaking after the middle of the subjective night. PER and TIM themselves form a heterodimer, interacting through PAS domains. The heterodimer moves to the nucleus and functions as the negative element in the feedback loop, acting on the positive regulators CLK and CYC to suppress their activation of the perltim promoters. This leads to a decline in the per and tim mRNA levels that continues throughout the night. The degradation of PER and TIM allows the cycle to start over.

The time delay between mRNA synthesis and the accumulation of PER and TIM is likely to be a critical element in the generation of the oscillation. PER is unstable in the absence of TIM. The dimerization stabilizes PER and promotes nuclear entry. In addition, both PER and TIM are phosphorylated, probably through the action of a homolog of casein kinase identified as double-time (dbt). This phosphorylation appears to be involved in regulation of PER turnover.

Mechanism of Entrainment

In Drosophila, light acts to cause a rapid decrease in the levels of TIM, and because TIM stabilizes PER, PER levels also decline. In the late day and early night when levels of these proteins are increasing, their destruction delays the progress of the oscillation, whereas in the late night and early day PER and TIM levels are decreasing, and hastening their demise advances the oscillation. Interestingly, genetic ablation of the eyes or mutations in the visual phototransduction pathway, although reducing sensitivity of the circadian clock to light, do not block its entrainment. The altered sensitivity to light observed with mutations that affect the visual system

FIGURE 3 Molecular model of the circadian pacemaker of Drosophila showing the proposed negative feedback loop of the oscillation. CLK and CYC heterodimers bind to E boxes of nuclear DNA promoting transcription of per and tim genes. TIM and PER proteins heterodimerize and are phosphorylated by DBT. The heterodimer enters the nucleus and inhibits the positive regulation by the CLK/CYC heterodimer. Light enters the system through CRY and promotes turnover of TIM and PER (modified from Dunlap, 1999).

indicates that an opsin-based photoreceptor can contribute to entrainment of the circadian rhythm of locomotor activity, but the persistence of entrainment in these mutants implicates an extraretinal photoreceptor. Action spectra for entrainment have suggested a flavin-based photoreceptor. Cryptochrome (CRY), is a member of a family of flavo-proteins, which includes photolyases and plant blue-light receptors. A mutant allele of the cry gene disrupts normal light responses of the locomotor activity rhythm, whereas flies overexpressing CRY are hypersensitive to light pulses. Further, in the periphery, CRY is required for light-dependent TIM degradation. These results suggest that CRY is a central element in the phototransduction pathway for entrainment.

The extent to which the molecular mechanisms detailed for Drosophila are applicable to other insects is not yet clear. However, there has been considerable progress in identifying homologous proteins in mammals, and although there are differences in detail, the basic framework of the oscillator seems to have persisted through the evolutionary process, giving confidence that the story will be broadly applicable to other insects as well.

Output of the Molecular Clock

The general supposition is that the clock ultimately regulates rhythms through the regulation of gene expression. This view is supported by the observation that there are several clock-controlled genes (CCGs) in Drosophila. However, at this point there are no examples in which a CCG has been linked directly to an overtly expressed physiological or behavioral rhythm, and this is an area of research that is likely to receive increased attention as researchers work to further elucidate the molecular details of the circadian system.

See Also the Following Articles

Brain and Optic Lobes • Drosophila melanogaster

Further Reading

Dunlap, J. C. (1999). Molecular bases for circadian clocks. Cell96, 271-290. Giebultowicz, J. M. (2000). Molecular mechanism and cellular distribution of insect circadian clocks. Annu. Rev. Entomol. 45, 769-793. Hall, J. C. (1995). Tripping along the trail to the molecular mechanisms of biological clocks. Trends Neurosci. 18, 230-240. Page, T. L. (1985). Clocks and circadian rhythms in insects. In "Sensory Physiology" (G. Kerkut and L. Gilbert, eds.), Vol. VI of "Comprehensive Insect Biochemistry, Physiology, and Pharmacology," pp. 577-652. Pergamon Press, Oxford. Page, T. L. (1990). Circadian organization in the cockroach. In "Cockroaches as Models for Neurobiology: Applications in Biomedical Research" (I. Huber, ed.), pp. 225-246. CRC Press, Boca Raton, FL. Plautz, J. D., Kaneko, M., Hall, J. C., and Kay, S. A. (1997). Independent photoreceptive circadian clocks throughout Drosophila. Science 278, 1632-1635.

Saunders, D. (1982). "Insect Clocks," 2nd ed. Pergamon Press, Oxford. Zitnan, D., Ross, L. S., Zitnanova, I., Hermesman, J. L., Gill, S. S., and Adams, M. E. (1999). Steroid induction of a peptide hormone gene leads to orchestration of a defined behavioral sequence. Neuron 23, 523-535.

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