News and Publications

Genetics and Genomics of Circadian Clocks

S.A. Kay, D. Alabadi, M.F. Ceriani, M. Covington, A. DeSchopke, S. Harmer, F. Harmon, L. Ho, T. Kuhlmann, P. Mas, S. Panda, R. Raman, T.F. Schultz, A. Scully, C. Strayer, K. Wager-Smith, F. Weber, M. Yanovsky

A vast array of cellular processes fluctuate with a 24-hour periodicity and occur at specific times of the day; these periodicities are known as circadian rhythms. The circadian biological clock generates these rhythms, which control diverse events ranging from the sleep-wake cycle in humans to the overall rate of photosynthesis in plants. Many pathologic changes in humans, such as sleep disorders, most likely are due to a defect in circadian rhythm, so understanding how the circadian clock operates within the cell is important for both plants and animals. The recent discovery of homologs to clock proteins between diverse species suggests that the elucidation of clock mechanisms in model systems will have broad impact on studies in humans. To study how circadian clocks are built inside cells, we use molecular and genetic approaches in 2 model systems: the plant Arabidopsis and the fruit fly Drosophila. We are also studying the genetics of sleep disorders in mice and humans.

In Arabidopsis, we used CAB2, a gene with circadian-regulated transcription, as a tool to dissect the circadian clock. This gene encodes a protein essential for photosynthesis, and its transcription peaks during the middle of the day and declines to basal levels at night. To study circadian-regulated transcription, we fused the clock-controlled CAB2 promoter to the firefly luciferase gene, which generates light. Transgenic plants containing this construct are imaged by using highly sensitive video cameras. With this method, we can measure gene expression noninvasively in living tissues, where rhythmic bioluminescence reflects clock control of transcription.

We screened mutants to look for seedlings that "glow" with an altered rhythm, and recently we used map-based cloning to identify the affected genes. These genes include TOC1, which encodes a putative clock component, a molecular cog that drives circadian rhythms, and the novel gene ZTL, which is involved in transmitting light signals from the environment to the circadian clock. Currently, we are elucidating feedback loops within the Arabidopsis circadian clock.

In another approach, we sought to determine the global pattern of gene regulation by the circadian clock in Arabidopsis. We used DNA chips to assay the expression pattern of approximately 8200 genes and found that more than 450 genes had circadian regulation. This study was the first of its kind and has produced a staggering array of genes for future studies of circadian clocks.

In Drosophila, we are interested in elucidating how circadian clocks are organized to control behavior and physiology in animals. Two key circadian clock genes were previously identified, period (per) and timeless (tim), and we cloned 2 new clock genes: dclock and dbMAL1. These 4 genes form an autoregulatory feedback loop of transcription, whereby the proteins dCLOCK and dbMAL1 promote transcription of per and tim and the proteins PER and TIM feedback to repress transcription of per and tim. Currently, we are investigating the mechanisms by which these 4 proteins interact to modulate gene expression.

To further identify proteins involved in the circadian clock, we generated transgenic Drosophila that express the luciferase gene driven by the per promoter. We developed an automated assay in which bioluminescence produced by live flies is monitored by using robotic plate-reading luminometers. We are using this automated bioluminescence system to isolate additional Drosophila clock mutants.

Animals with mutations in their clock genes have a distinctive pattern of behavior such that their active and restful periods are shifted either earlier or later in the day. A family of human sleep disorders results in similar phenomena, and recently researchers found that a clock gene is responsible for a familial form of a sleep disorder. We are doing a human genetics study to determine the role of circadian clock genes in human sleep disorders. We are also screening mice for abnormal daily behavioral rhythms with the goal of identifying novel circadian mutants. With this approach, we hope to develop preclinical models of human sleep disorders and to further explore the link between mouse and human circadian dysrhythmias.

PUBLICATIONS

Covington, M.F., Panda, S., Lui, X.L., Strayer, C.A., Kay, S.A. ELF3 modulates resetting of the circadian clock in Arabidopsis. Plant Cell 13:1305, 2001.

Darlington, T.K., Lyons, L.C., Hardin, P.E., Kay, S.A. The period E-box is sufficient to drive circadian oscillation of transcription in vivo. J. Biol. Rhythms 15:462, 2000.

Devlin, P.F., Kay, S.A. Cryptochromes are required for phytochrome signaling to the circadian clock but not for rhythmicity. Plant Cell 12:2499, 2000.

Devlin, P.F., Kay, S.A. Flower arranging in Arabidopsis. Science 288:1600, 2000.

Harmer, S.L., Hogenesch, J.B., Straume, M., Chang, H.S., Han, B., Zhu, T., Wang, X., Kreps, J.A., Kay, S.A. Orchestrated transcription of key pathways in Arabidopsis by the circadian clock. Science 290:2110, 2000.

Kreps, J.A., Muramatsu, T., Furuya, M., Kay S.A. Fluorescent differential display identifies circadian clock-regulated genes in Arabidopsis thaliana. J. Biol. Rhythms 15:208, 2000.

Lyons, L.C., Darlington, T.K., Hao, H., Houl, J., Kay, S.A. Specific sequences outside the E-box are required for proper per expression and behavioral rescue. J. Biol. Rhythms 15:472, 2000.

Mas, P., Devlin, P.F., Panda, S., Kay, S.A. Functional interaction of phytochrome B and cryptochrome 2. Nature 408:207, 2000.

Strayer, C., Oyama, T., Schultz, T.F., Raman, R., Somers, D.E., Mas, P., Panda, S., Kreps, J.A., Kay, S.A. Cloning of the Arabidopsis clock gene TOC1, an autoregulatory response regulator homolog. Science 289:768, 2000.

Wager-Smith, K., Kay, S.A. Circadian rhythm genetics: From flies to mice to humans. Nat. Genet. 26:23, 2000.

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