Circadian Rhythm Homology and Divergence

Courtesy of ScienceThe fruit fly circadian cycle shares three strong similarities with the mammalian: the per gene itself, CLOCK and BMAL regulation of per, and a gene called tau in hamsters and double-time (dbt) in flies, both of which encode the enzyme CKI*. Molecular biologists have been teasing apart the intricate innards of organisms' biological clocks for decades, gaining rare insight into a veritable bridge between genes and behavior. Those clocks' circadian rhythms, the 24-hour cycles that govern physiological and metabolic functions, enable organisms to follow the outside world's cues of light and dark. A better understanding of circadian rhythms promises to shed light on the mechanics of sleeping disorders, help optimize drug delivery, help night shifters improve alertness, and elucidate the anatomy of seasonal affective disorders and manic depressive illness. Potential applications, though, extend beyond humans to plants and animals whose photoperiodism might one day be adjustable so that they seed or breed according to farmers' needs. "[Circadian rhythms] touch every aspect of biology," comments Jay Dunlap, chairman of genetics at Dartmouth Medical School and an investigator into the circadian rhythms of mammals and the bread mold Neurospora, one of the first studied circadian rhythm models. Interest in circadian rhythms is due not only to their practical applications. They also have an intrinsic appeal as an unparalleled window on the phylogeny of several animals. Thus far, homology among animals, cyanobacteria, fungi, and plants appears relatively limited, suggesting that each has evolved independently (though Dunlap recently showed Neurospora homology with a mammalian clock gene called bmal, and plants are known to have homology with animal genes called cryptochromes). But investigators have uncovered a remarkable and fascinating degree of conservation among the molecular players driving the clocks in animals, from fruit flies to humans. Yet, even here, as researchers continue to explore the more complex mammalian system, significant areas of divergence, perhaps most notably between the oft-compared mouse and fruit fly, are starting to emerge--especially with regard to the coordination between cellular clocks in the brain and the rest of the body. "The surprising thing is that in terms of the animal kingdom, we haven't yet gotten into an area where we're seeing a divergence in the pieces [of the clock] in the sense of the nature of the genes involved," suggests Michael Young, head of the laboratory of genetics at Rockefeller University. "The kinds of things we're seeing instead are the same genes but used in different ways within this mechanism." An orchestra of similar molecular players conducts the daily rhythms in, for example, Drosophila and mammals. In the fruit fly, bmal (also called cycle) and clock start the cycle by producing a complex of CLOCK and BMAL protein products, which activates transcription by the genes period (per) and timeless (tim) to produce PER and TIM. As part of a negative feedback loop, the PER-TIM complex moves to the nucleus, where--like clockwork--it suppresses the CLOCK/ BMAL complex, thus preventing further transcription from per and tim. The fruit fly circadian cycle shares three strong similarities with the mammalian: the per gene itself, CLOCK and BMAL regulation of per, and a gene called tau in hamsters and double-time (dbt) in flies.1 Researchers at Northwestern University led by Howard Hughes Medical Institute investigator Joseph Takahashi, a professor of neurobiology and physiology, shocked the field when they showed that tau produces the same enzyme, casein kinase I* (CKI*), as dbt. In both cases, the genes are believed to make protein products that help the circadian cycle along by controlling the accumulation of PER and encouraging its degradation. Although CKI* has been known to play a central role in fruit fly circadian rhythms, no one expected that the tau mutation, first isolated in the Syrian hamster 12 years ago,2 would encode the same protein as the fruit fly's dbt. "The fact that you're pulling the same genes out of the mouse and the fly suggests that the small collection of genes that you have in the fly ... [includes] most of the potent contributors to this biology," comments Young. Timeless Questions Investigators are likely to find more players in mammals, already known to have two additional per genes, a possibly more complicated role for tim, and probably a different role for cryptochromes. Whereas in mammals, the two known cryptochromes, Cry1 and Cry2, appear to be transcriptional regulators that are gears of the clock machinery itself and have no role in absorbing and processing light, the only known cryptochrome gene in Drosophila seems to be integral to the organism's light processing, something like the known cryptochromes in plants. A recent study by Michael Rosbash, a Howard Hughes Medical Institute investigator and professor of biology at Brandeis University, suggests that cry is indeed the only photoreceptor in flies through which light regulates rhythms.3 Under constant light, normal flies go into arrhythmia. So-called cryb mutants did not, however, suggesting that cry is the lone light gatekeeper. Rosbash cautions, though, that, unlike in mammals, such studies of Drosophila are based on single mutants, like cryb, in which the target function may not have been completely erased. Mammalian knockout models, though their phenotypes may be tough to interpret, don't have that problem. It's a caveat, says Rosbash, that makes difficult a definitive analysis of cryptochrome function in mice versus flies. Though most circadian investigators believe that cryptochrome function has diverged significantly, Rosbash suggests that conclusions are still premature. In mammals, cryptochromes appear to have actually fulfilled the role of tim in flies. They help transport PER into the nucleus, and they're involved in negative regulation of the CLOCK/BMAL complex.4 To explain and illustrate the implications of this divergence of function, a team led by Steven Reppert, a professor of pediatrics at Harvard Medical School, recently reported evidence that intricate interacting positive and negative feedback loops--involving the known molecular players--exist in the mouse, and they work in coordination to reinforce 24-hour oscillations.5 Investigators won't know how important these loops are for generating a rhythm until all or parts of them have been eliminated and the resulting phenotypes studied. Says Takahashi, "It's there, but now the issue is [determining] its real role." A definitive analysis of tim in mice (called mTim) has also proved difficult. In late 1998, a group of researchers reportedly found the true mouse homologue of Drosophila tim.6 But Reppert's group expressed skepticism, saying that the gene isn't rhythmic at the protein or the RNA level in the suprachiasmatic nucleus (SCN), the master clock in the mammalian brain; that it's not light regulated; and that it has no influence on moving PER into the nucleus.4,7 Adding to the skepticism, the recently completed sequencing of the Drosophila genome revealed a second timeless-like gene far away from the known clock-relevant tim. The new Drosophila gene appears to be much more similar to mTim than to the original fruit fly gene. Interestingly, the high degree of homology between flies and mammals may have made investigators a bit too anxious to fit mTim into the growing circadian puzzle of molecular players. "We've all been barking up the wrong circadian tree," says Reppert. "Some people had a lot invested in that being a real homologue," maintains Dunlap. "And it's pretty clear it's not." Young, though, points to evidence that, in cultured cells, TIM will regulate mPer transcription and, in the mouse brain, will bind to CRY. Such results suggest that mTim has a role of some sort in mammalian clocks, though perhaps one significantly different from that in flies. Takahashi is withholding judgment until there's evidence for a second timeless in mice. Further exploration of the function of the second Drosophila timeless gene should also shed some light on the issue. Michael Young Whole-body Oscillations Details aside, the molecular homology between the fly and mammal is no doubt stunning. Similarities on a "systems wide" level, however, remain uncertain. Although investigators have known for some time that circadian oscillators exist throughout the bodies of several animals (not to mention plants), researchers are only now starting to understand how the well-characterized mammalian SCN cooperates with the body's peripheral clocks. Lateral neurons in the fly are only partially analogous; they appear to be the source for the timing of locomotor activity behavior but don't seem to communicate with peripheral oscillators. Takahashi compares the relationship between the SCN and peripheral oscillators in the mammal to that between the heart and cardiac cells. Like the heart, it's a hierarchical system driven by a pacemaker (the SCN); peripheral clocks, like cardiac cells, have the capacity to oscillate independently but still depend on the SCN. While mammals have photoreceptors for entraining circadian rhythms exclusively in the eye, animals such as fruit flies, birds, lizards, and fish have distributed photoreceptors often in directly light- sensitive peripheral organs. In the case of the latter, the clocks are easily "resettable" when the animals undergo a phase shift. Internal mammalian clocks, however, can apparently more easily be nudged out of sync with each other. A recent paper by Michael Menaker, a professor of biology at the University of Virginia, helped clarify the SCN-peripheral oscillator relationship in rats.8 A day after putting the rats through six-hour phase shifts, they sacrificed them and looked for oscillation peaks in various tissues. The SCN adjusted almost immediately, but peripheral structures' phase shifted very slowly. "The SCN's really self-sustaining," Menaker surmises. "It can go 30 cycles for a very long time, especially in culture. Peripheral oscillators have the capacity to have an oscillation, but they're not self-sustained in the absence of the body." By the time the SCN had shifted completely, some peripheral oscillators had also shifted, and others had not--the liver, for example, takes a particularly long two weeks to catch up with the rest of the system. These results lend strong molecular evidence to the long-held hypothesis explaining the symptoms of jet lag sufferers and night shift workers who have a hard time adjusting to phase changes: The human body is experiencing internal desynchrony. According to Menaker, if scientists could identify the signals that put the SCN in touch with peripheral oscillators, they might be able to fashion a treatment by manipulating those signals. Although scientists guess the signals could involve hormones, the actual mode of communication remains a mystery and continues to be the focus of a hot area of circadian research. "At the molecular level, which is the level of generating the oscillation within the cell, there are a lot of homologies," explains Menaker. "There are some differences, but the basic system has been apparently conserved for 600 million years." At the "systems" level, says Menaker, although there are some similarities, the divergence is more significant. "And that's not surprising either, when you think about the fact that these are really very different animals and they have very different things to cope with in this environment." S Eugene Russo can be contacted at erusso@the-scientist.com. References 1. P.L. Lowrey et al., "Positional syntenic cloning and functional characterization of the mammalian circadian mutation tau," Science, 288:483-91, April 21, 2000. 2. M.R. Ralph, M. Menaker, "A mutation of the circadian system in golden hamsters," Science, 241:1225-7, 1988. 3. P. Emery et al., "Drosophila cryptochromes: a unique circadian-rhythm photoreceptor," Nature, 404:456-7, March 30, 2000. 4. K. Kume et al., "mCRY1 and mCRY2 are essential components of the negative limb of the circadian clock feedback loop," Cell, 98:193-205, 1999. 5. L.P. Shearman et al., "Interacting molecular loops in the mammalian circadian clock," Science, 288:1013-9, May 12, 2000. 6. A.M. Sangoram et al., "Mammalian circadian autoregulatory loop: a timeless ortholog and mPer1 interact and negatively regulate CLOCK-BMAL1-induced transcription," Neuron, 21:1101-13, November 1998. 7. M.J. Zylka et al., "Molecular analysis of mammalian timeless," Neuron, 21:1115-22, 1998. 8. S. Yamazaki et al., "Resetting central and peripheral circadian oscillators in transgenic rats," Science, 288:682-5, April 28, 2000.

Circadian Rhythm Homology and Divergence

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