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Beyond Insomnia: Strategies of Circadian Genomics
Edward R. Winstead

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With people flying across time zones in ever-increasing numbers, jet lag is attracting lots of attention. In fact, research on biological rhythms is now a hot area in genomics. Achievements such as the identification of a core set of genes underlying biological rhythms have attracted new talent to the field, and laboratories keep churning out studies that may have relevance beyond understanding the cycles of sleeping and waking.

Courtesy of Sharon Low-Zeddies
These chimeric mice have both normal cells and cells with the Clock mutation.

"This field is hot because circadian rhythms have been cracked open in the last four years," says Joseph S. Takahashi, a Howard Hughes Medical Institute investigator at Northwestern University. Today it is clear that the same basic genes are involved across species in generating daily rhythms. The sequencing of the human, mouse, fly and worm genomes confirms the evolutionary importance of these genes. For instance, important mammalian 'clock' genes have turned up in the fly, and clock genes from Drosophila are present in humans.

The strategies used to discover circadian genes and link them to behavior are perhaps as significant as what was discovered. "My colleagues in mouse genetics or genomics are more interested in that aspect of the work than in the circadian rhythms," says Takahashi.

‘The first case of a single mutation that completely abolishes the circadian clock’

In 1994, Takahashi led a mutagenesis screen for 'clock' mutants and identified a mouse with an abnormal daily rhythm, or period length. Three years later, his team cloned the particularly large gene, named Clock, using a combination of techniques, including shotgun sequencing.

"The genomic sequence was important when we were cloning the gene, because that was the best way to find it," says Takahashi. After his laboratory geared up for the initial shotgun sequencing of Clock, he became taken with the technology and later decided to finish the job. "There is not that much finished mouse sequence," he noted at the time. (Celera has recently announced the assembly of mouse.)

In December, his laboratory published the finished and annotated genome sequence in Genome Research. The paper includes a comparison between the Clock locus and its counterpart on human chromosome 4. "A nice thing happened in that the human sequence was available," says Takahashi.

Physical map of the 204 kb flanking the Clock locus. View larger

"Now we have everything," says Lisa Wilsbacher, a member of the sequencing team. "This is for now one of the largest regions of contiguous mouse sequence, and it is fully annotated. We have the raw material to do functional studies."

The analysis revealed two previously unidentified mouse genes near Clock, as well as sequences found in both humans and mice that may be regulatory elements. The regulation of Clock is of interest to Takahashi's group because they found that mice with additional copies of the gene had decreased period lengths. Thus, too much expression of Clock may be disruptive to circadian rhythms.

"We are very interested in how the Clock gene is regulated," says Wilsbacher. "The conserved sequences between humans and mice give us some targets for possible regulatory regions."

Models of biological clocks suggest that the core mechanisms are 'feedback loops' involving interactions between genes and proteins; these interactions somehow trigger appropriate fluctuations in gene expression and keep the cycling going. But what exactly drives the tightly regulated expression of these genes is still being worked out.

In the June issue of the Journal of Neurobiology, researchers show that a genomic sequence of six nucleotides in Drosophila plays a role in activating one of the major clock genes, timeless. The sequence, known as an E-box, resides in the promoter region of timeless, and is the binding target of two circadian proteins. The proteins—dCLOCK and CYCLE—come together and bind to the E-box, thereby activating the timeless gene.

"The basic idea is that these proteins bind to the E-box at certain times of day and not others, and that's what makes the gene cycle," says Amita Sehgal, of University of Pennsylvania Medical School, who led the study. The new data are consistent with a more complex version of this model. "Clearly the E-box is important but there could be multiple E-boxes and other sequences that might play a role in the cycling," she adds.

Schematic representation of the tim transgenic constructs and of the oligonucleotide used for Electrophoretic Mobility Shift Assays (EMSAs). View larger

The researchers restored circadian rhythms in flies lacking timeless by inserting a new copy of the gene and its promoter. The 'therapy' failed, however, when the researchers inserted the gene but also mutated the E-box.

The importance of E-boxes in circadian genes has been suggested by work in cultured cells. The Pennsylvania study is one of the first papers on E-boxes in actual flies. In February, researchers at Brandies University in Waltham, Massachusetts, reported that a normal circadian rhythm in flies depended on closely spaced E-boxes in the promoter region of timeless. The research was reported in Molecular Cell Biology.

In a third study—this time in mice—researchers at the University of Wisconsin Medical School in Madison showed that the Mop3 gene is important in regulating the cycling of the mouse clock. Christopher A. Bradfield and colleagues hypothesized that the MOP3 protein is the partner of the CLOCK protein, and they confirmed its circadian role by knocking the gene out in mice: Animals without Mop3 had no circadian rhythms.

"This is the first case of a single mutation that completely abolishes the circadian clock," says Bradfield, who collaborated with Takahashi's laboratory on the study. The findings were published in a December issue of Cell.

The core of biological clocks are ‘feedback loops’ of interacting genes and proteins

"We view the MOP3 and CLOCK proteins as engines that drive the expression of circadian genes," says Bradfield. As in the Drosophila clock, the two proteins appear to form a complex that binds to a regulatory sequence in clock genes. The E-box (5'-CACGTG-3') promoter element/regulatory sequence is identical in flies and mice.

At Northwestern and the University of Wisconsin, researchers measured the effects of the missing protein on mouse cells and on the animal's behavior. The mice lacked biological rhythms, and for reasons that are not clear, the animals were less active than normal. At the molecular level, the consequences of losing the protein were evident in the brain as well as in peripheral clocks. "Liver clocks were completely disrupted," says Bradfield.

The researchers concluded in the Cell paper that the broad consequences for the knockout mice suggest that "MOP3 rests near the top of the circadian gene hierarchy in mammals."

"The clock story has become much clearer recently, and we seem to have the main players," says Bradfield. "But no one has been able to put the pieces together and show how this complex scheme turns into an actual 24-hour clock."

Not long after the discovery of the Clock mutant, a member of Takahashi's laboratory, Sharon S. Low-Zeddies, began an analysis of the effects of the Clock mutation on the circadian behavior using chimeric mice. The study was designed to explore functional and physiological questions and to further characterize the effects of the Clock mutation on behavior.

Reporting their findings recently in Cell, the two researchers describe circadian behavior in the chimeric mice as the result of the integration of genetically distinct cells. Chimeric animals have the equivalent of two genomes in one body, and the analysis considered the effects of including normal and mutant circadian cells in the same tissues.

"The focus in the circadian field has been on genes and gene discovery, and that has given us a description of the conserved mechanisms of circadian clocks in animals," says Takahashi. "The chimera study is a completely different level of analysis from what we've seen over the last four years. It shows that while genes are important, you also have to understand how cells integrate information."

The study was possible because one place in the brain is the master control of circadian rhythms in mammals. The central clock mechanism is localized in a defined region of the hypothalamus called the suprachiasmatic nuclei, or SCN. By contrast, learning and memory involve more complex circuits of neurons.

Researchers have known for 30 years that the SCN is the central pacemaker, but little else about the region is known. "With the chimeric mice, we tried to figure out how SCN cells talk to each other to produce a coherent circadian rhythm in the whole body," says Low-Zeddies.

Chimeras exist only in laboratories and cannot be produced through mating. Low-Zeddies created the animals by combining two different mouse embryos early in development: Each embryo had a genome that differed at the CLOCK locus—one with a mutant gene and the other with a normal gene.

The resulting animal had two distinct types of cells throughout its body in different ratios in different tissues. The SCN, for example, might be 60 percent mutant cells and 40 percent normal cells—or any other combination. With chimeric animals, it is possible to get anywhere from 0 to 100 percent of mutant cells throughout the body, and Low-Zeddies developed an efficient system for measuring the ratios.

The SCN is a discrete group of cells that controls a well-characterized behavior; by creating animals with different combinations of normal and mutant cells in the SCN, the researchers could see quantitative associations between different cell types and behavior. "For a neuroscientist, this is a dream come true," says Low-Zeddies.

The circadian behavior of chimeras correlated with the overall composition of SCN cells. Animals with mostly mutant cells had mutant-like circadian behavior; animals with mostly normal cells behaved normally. "There is a complex integration among cells in the brain that requires a majority of cells of one genotype to dominate the behavior," explains Low-Zeddies.

When there was a rough balance between normal and mutant cells, the result was a variety of 'intermediate' circadian behavioral profiles that no one had ever seen. Furthermore, within the same animal the researchers saw behavior that is characteristic of both normal and mutant mice. "It seemed that different groups of cells were somehow taking control at different times," she says.

The findings suggested that two clock-related effects long associated with each other may actually involve different biological processes. Clock mutants often have abnormal cycle length (period) and abnormal activity (amplitude), but some chimeras had normal period but abnormal amplitude or the reverse. This suggested to the researchers a greater complexity than generally assumed.

"We conclude that the effects of the Clock mutation on period and on amplitude were separable at a cellular level," says Low-Zeddies. "That is, they may be determined by non-identical groups of cells within the SCN."

Takahashi hints that interesting research on circadian rhythms and human health is coming. "We are still trying to document the ways that circadian rhythms are important," he says. "If you lose your clock you don't die, but I think there will be information in the future that says you are compromised."

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Wang, G.K. et al. Regulation of the cycling of timeless (tim) RNA. J Neurobiol 47, 161-175 (June 2001).
Low-Zeddies, S.S. & Takahashi, J.S. Chimera analysis of the Clock mutation in mice shows that complex cellular integration determines circadian behavior. Cell 105, 25-42 (April 6, 2001).
McDonald, M.J., Rosbash, M. & Emery, P. Wild-type circadian rhythmicity is dependent on closely spaced E boxes in the Drosophila timeless promoter. Mol Cell Biol 21, 1207-1217 (February 2001).
Bunger, M.K. et al. Mop3 is an essential component of the master circadian pacemaker in mammals. Cell 103, 1009-1017 (December 22, 2000).
Wilsbacher, L.D. et al. The mouse Clock locus: sequence and comparative analysis of 204 kb from mouse chromosome 5. Genome Res 10, 1928-1940 (December 2000).

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