New "Clock Gene" Uncovered
Genome-Wide Approach Yields Result

By Jason Socrates Bardi

The solving of the human genome sequence was hailed a few years ago as biology's equivalent to landing a man on the moon—a mammoth milestone of monumental importance.

However, unlike the first moon shot, the real milestone of the human genome project is not a singular event. The genome project's giant leap for mankind is coming not with a single small step taken on one summer's night but with thousands of small steps spread out over the course of several years.

The importance of solving the human genome and the genomes of other species is that those billions of letters of DNA are deposited into databases and become available to scientists everywhere to conduct post-genomic research. Such research includes annotating the human genome—matching the DNA codes that hold the secret to human life to the genes, proteins, physiologies, and behaviors that define human life. Such work holds great promise for future medicine, and scientists have been investigating how the genes in the human genome actually contribute to the biology of health and disease.

In the last few years, a team of scientists from The Scripps Research Institute and the Genomics Institute of the Novartis Research Foundation (GNF) has been working toward an understanding the biology of circadian rhythms—the cyclic, clock-like expression of genes in the body.

The team, led by Steve Kay and John Hogenesch uses a combination of genomic, biochemical, and behavioral approaches, work that recently revealed a new genetic component of the mammalian clock—a protein known as "Rora."

This discovery may someday help people with jet lag, shift workers who feel wiped out after working a night shift, and people with more serious sleep disorders, many of which are related to circadian rhythms, say the scientists, who report their findings in the latest issue of the journal Neuron.

In addition to Kay and Hogenesch, authors on the paper include Satchidananda Panda, a graduate of Scripps Research's Kellogg School of Science and Technology and currently a postdoctoral fellow at GNF, and Trey Sato, currently a postdoctoral fellow at the Scripps Research La Jolla campus who will arrive at Scripps Florida as a staff scientist in December. Kay is a professor of cell biology and the director of the Institute for Childhood and Neglected Diseases at The Scripps Research Institute. Hogenesch is currently a researcher at GNF and will soon become an associate professor and the head of Genome Technology at Scripps Florida.

Circadian Rhythms and Jet Leg: Blame the Liver

Science has known for years that humans, mice, and many other plants and animals possess internal clocks that keep track of time and coordinate physiological, behavioral, and biochemical processes with the rhythm of the 24-hour cycle of day and night.

These so-called circadian rhythms offer distinct advantages to organisms that use them. Plants, for example, shut down photosynthesis at night, and they gear up their photosynthetic machinery and raise their leaves just before dawn. They use their clocks to measure day length and in that way anticipate changes in the seasons—a system that determines when they shed their leaves, produce seeds, and make flowers or fruit.

Humans also have circadian rhythms, and we entrain our internal clocks to the 24-hour day. Under normal conditions, we time our major activities with daylight, we sleep during the nighttime, and some of our vital signs follow this pattern. Our blood pressure fluctuates daily, rising and falling at predictable times of day and night.

Scientists have provided evidence of the existence of internal clock mechanisms by placing organisms like rodents in chambers isolated from day/night cycles. In spite of this, the animals' rhythms still cycle approximately every 24 hours.

"Even if you were to put the lights out on us, we would still [time] our activity to when our body expects light," says Hogenesch.

One of the most intriguing aspects of this is that the mammalian clock is actually composed of many separate clocks that maintain different circadian rhythms specifically adapted to the various tissues of the body.

The liver, the heart, and the kidneys each have their own distinct clocks. The liver, for instance, expresses a number of enzymes that remove toxic substances from the bloodstream during the day, which corresponds with the prime time for food (and toxic compound) intake.

Coordinating the activities of all these different clocks is the job of the master circadian oscillator, or master clock, which in humans and other mammals is the suprachiasmatic nuclei, a small center in the brain's hypothalamus with about 10,000 neurons that sits above the optic chasm—the location where the optic nerves cross each other.

This master clock synchronizes independent clocks that reside in peripheral tissues, and every 24 hours, the master clock cycles. This cycling involves the coordinated expression of many genes involved in feedback loops, in which the expression of one gene turns on the expression of a second gene, which turns off the first gene, which turns off the second gene, which turns the first gene back on, etc., day in and day out.

However, in real life, the situation is not so simple as a loop involving only two genes. Multiple clock genes in mammals are involved in overlapping feedback loops. The clock also keeps time dynamically, constantly shifting to stay in time with environmental changes. And these adjustments vary from tissue to tissue. Different tissues respond to the clock in their own way, and they all reset their own clocks independently of one another.

The heart, for instance, is like an obsessive-compulsive clock watcher. It monitors the master clock closely and trains its circadian rhythms to changes in the master clock rapidly.

The liver, on the other hand, is more slothlike. It is less attentive to the master clock, and it takes several days for the liver to catch up with changes in the master clock. Incidentally, the problems related to jet lag and night shift work are often caused by the liver's inability to respond rapidly to changes in the sleep-wake cycle.

One key to understanding the intricacies of the mammalian clock and to addressing problems with jet lag and certain sleep disorders is discovering the different genes that communicate the timing of the master clock with the circadian rhythms of the various tissues. Not all of these genes are known.

Identifying the Clock Genes

Panda and Sato performed gene array experiments on different samples of mammalian tissue to determine which genes are cycling and which might be components of the clock.

These experiments involve taking cells from the particular tissues, recovering the cells' expressed genes (in the form of messenger RNA, or mRNA), chopping the mRNA into fragments, and plopping the mixture of fragmented mRNA on a gene chip— a glass or silicon wafer that has thousands of short pieces of RNA attached to it with sequences corresponding to known genes.

These short pieces are laid out in a grid, and genes that are expressed in the tissue will bind to complementary pieces of mRNA on the grid. Then by looking to see which pieces on the grid have RNA bound to them, the scientists are able to determine which genes were being expressed in the sample.

DNA and RNA chips have become a standard tool for genomics research in the last couple of years, and scientists can quite easily put a large number of different oligonucleotide pieces—conceivably even all the known genes in an organism—on a single chip.

The Scripps Research and GNF team charted the time course of circadian rhythms by looking at the expression of 10,000 different genes in various murine tissues every three hours over the course of two days. They examined the gene expression in the liver, kidney, aorta, skeletal muscle, and the suprachiasmatic nucleus of the hypothalamus, and they looked for cyclic expression.

What the data showed was that about 10 percent of the genes cycled, but most showed little overlap from tissue to tissue. This type of cycling has to do with local physiology—for instance, the liver's expression of certain enzymes at certain times of the day. What they were really interested in were the overlapping cyclers—those genes that cycled in all tissue types. These, they reckoned, would be part of the master clock.

They found 50 genes that cycled at the same time throughout the day across all the various tissues, and they speculated that this collection would include both known and unknown circadian rhythm genes. Indeed, known circadian genes were among the 50, but there were dozens of other cycling genes that had not been previously identified as clock genes.

The scientists speculated that some of these other genes may be part of the mammalian circadian clock, and they reasoned that if they were, they might interact with some of the known clock genes. So they designed an experiment to see if expressing the new genes in a cell could change the expression of known clock genes. To do this, they used a biochemical assay to detect if any of these genes had the ability to control transcription—that crucial first step in the expression of a gene.

They discovered that a family of genes called the retinoic acid receptor-related orphan receptor-a (Rora) cycled and had the ability to control transcription. These are so named because they are similar in amino acid sequence to the retinoic acid receptor genes, although they do not have the same function and do not bind to retinoic acid. (They are called orphan receptors because it is not known what activates them).

The Flick of a Switch

Rora is a gene that produces a transcription factor—a type of regulatory protein that binds to DNA and can turn gene expression on or off like the flicking of a switch.

Rora, once it is turned on, activates the transcription of a gene that encodes another transcription activator known as Bmal-1, which is one of the known circadian genes. Bmal-1 drives the transcription of a protein called cryptochrome, which subsequently inhibits the ability of Bmal-1 to activate cryptochrome's own transcription. This feedback loop is what keeps the body entrained to a 24-hour day.

Since Bmal-1 is so crucial to keeping the body's clock entrained, finding something like Rora, which alters Bmal-1's expression, is significant and suggests that Rora is also part of the mammalian clock. But the scientists wanted to go further and prove that Rora protein plays a role in the circadian rhythms inside a living creature.

They observed a mutant murine model that has a defective Rora gene. This murine model is called "staggerer" because its genetic defect causes a characteristic loss of coordination.

As it turns out, the staggerer model with a defective Rora gene also has a defect in its ability to regulate its circadian clock. The team of researchers showed that staggerers have aberrant circadian rhythyms and a shortened clock that is only 23.2 hours long. This situation is sort of like a grandfather clock that cannot keep good time and runs too fast because it has a faulty spring balance.

"What we are showing is that circadian clocks are composed of interlocking feedback loops," says Kay. The overlapping feedback, says Kay, is probably there for a number of reasons. It makes the clock more robust and resilient to change. It means that there is more than one cycle in which changes to clock genes can affect changes to other genes, and therefore the clock can be reset more easily.

Knowing that Rora is a component of the mammalian clock is significant because it may be a valuable target for the development of compounds to correct sleep disorders, many of which are related to circadian rhythms, and for countering the most common circadian problems—the jet-lag one feels after overseas flights or fatigue when working night shifts.

"Identifying a ligand for Rora might help reduce the effects of jet lag," says Sato.

The article, "A Functional Genomics Strategy Reveals Rora as a Component of the Mammalian Circadian Clock" was authored by Trey K. Sato, Satchidananda Panda, Loren J. Miraglia, Teresa M. Reyes, Radu D. Rudic, Peter McNamara, Kinnery A. Naik, Garret A. FitzGerald, Steve A. Kay, and John B. Hogenesch and appears in the August 19, 2004 issue of the journal Neuron.

Send comments to: jasonb@scripps.edu

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Professor Steve Kay (above) conducted research with John Hogenesch and other colleagues that revealed a new genetic component of the mammalian clock—a protein known as "Rora."