Insight into how the body tells time

You may feel different at the dreary hour of 4 a.m. than you do mid-afternoon at 4 p.m. Now, researchers might understand why. A study from Washington University School of Medicine in St. Louis helps explain how genes dictate our biological clock.

Nearly all living things have a natural rhythm that influences their behavior and physiology. This rhythm typically is “circadian”, following a near 24-hour cycle. Driven by an internal clock, a creature’s natural rhythm is synchronized to the outside world by external cues, like the sun. So far, the products of eight different genes have been discovered to be essential to the operations of this clock. Scientists believe that these genes, in turn, somehow influence the expression of other genes throughout the body in order to control the timing of behaviors like sleep and wakefulness.

Researchers from three laboratories at the School of Medicine, in collaboration with a team at Affymetrix, have identified 22 genes that appear to be rhythmically regulated by the internal clock of the Drosophila fly and found hundreds more genes that are regulated by both light and the internal clock. The study appears in the June 24 issue of the Proceedings of the National Academy of Sciences.

“Understanding how our internal environment responds to our innate biological clock could help us develop better ways of adjusting to challenging circumstances, like unusual work shifts or jet lag following a long journey,” says lead investigator Paul H. Taghert, Ph.D., professor of anatomy and neurobiology.

The fruit fly Drosophila melanogaster is one of the most commonly studied organisms, particularly in the pursuit of understanding biological clocks. In the past, researchers only could estimate the number of genes affected by the eight clock genes. But now that the fly’s genome has been fully sequenced, scientists can scrutinize nearly all of the animal’s 14,000 genes.

The Washington University team capitalized on the genome database now available. Using a relatively new technology called DNA microarrays – comprehensive lists of all the active genes in a tissue sample – they measured the expression levels of nearly 14,000 genes at various time-points in the heads of normal flies and in flies missing one of the clock genes, called period.

All flies were exposed to light for 12 hours, followed by dark for 12 hours. The cycle continued for a total of 96 hours. Genetic analyses were performed on half of the flies at six different time-points on the fifth day.

The remaining flies were transferred into complete darkness for 48 hours. On the third day of darkness, the team again analyzed gene expression at each of six time-points. By exposing flies to constant darkness, the team hoped to detect genetic changes that are regulated by the internal circadian timekeeping system, rather than by external cues.

Overall, the researchers obtained over 70 readings for each of the nearly 14,000 genes, generating about a million individual measurements.

Using sophisticated computer-based statistical analyses, the team determined that between 72 and 200 of the flies’ 14,000 genes showed significant rhythms of gene expression in normal flies living in a daily light-dark cycle. Of these 72 genes, 22 continued to fluctuate when flies were collected after three days of complete darkness. This implies that these 22 genes are driven by the internal, circadian clock, not by external cues such as light.

Mutant flies lacking the period gene also were placed into the same two experimental conditions – light and dark fluctuations compared with complete darkness. The flies exposed to alternating light and dark still showed 18 genes with persistent, rhythmic oscillations, demonstrating that light and dark can directly drive rhythmic gene expression.

The remaining 32 of the 72 oscillating genes only fluctuated rhythmically in animals that still had the period gene and who were exposed to light and dark conditions. The biologic functions of most of these oscillating genes are unknown.

One of the most surprising results of the study was the discovery of hundreds of genes whose levels did not fluctuate with time of day, but responded drastically to different lighting conditions or to the presence or absence of a circadian clock.

“The fly’s ability to regulate a large fraction of all its genes depending on a combination of day length and the circadian clock gives us an attractive model for understanding seasonal timekeeping,” says co-author Russell N. Van Gelder, M.D., Ph.D., assistant professor of ophthalmology and visual sciences. “Defects in seasonal timekeeping are thought to be related to seasonal affective disorder (SAD), in which individuals experience recurrent depression during the short days of winter.”

Three similar studies were published immediately preceding this paper, each estimating the number of genes controlled by the internal clock to be more than 100. Eighteen of the 22 genes identified in this study also were identified by one of the other three studies. However, the majority (84 percent) of the remaining genes identified by the other three groups were not included in any of the other lists.

“We feel that our analysis provides a minimal set of circadian genes about which we can feel fairly confident,” says Taghert.

In an effort to optimize research initiatives, the School of Medicine team has posted all of their raw data on the Internet at http://circadian.wustl.edu.

Lin Y, Han M, Shimada B, Wang L, Gibler TM, Amarakone A, Awad TA, Stormo GD, Van Gelder RN, Taghert PH. Influence of the period-dependent circadian clock on diurnal, circadian, and aperiodic gene expression in Drosophila melanogaster. Proceedings of the National Academy of Sciences, June 24, 2002.

Funding from the National Institutes of Health, the Medical Scientist Training Program, the Research to Prevent Blindness Career Development Award, the Becker/AUPO/RPB Clinician-Scientist Award and from the Human Frontier Science Program Organization supported this research.