News and Publications

Genetics and Genomics of Circadian Clocks

S.A. Kay, M. Covington, A. DeSchopke, E. Farre, S. Harmer, F.G. Harmon, L. Ho, T. Imaizumi, R.M. Leiber, P. Mas, S. Panda, R. Raman, T.K. Sato, T.F. Schultz, H.G. Tran, K. Wager-Smith, D.K. Welsh, F.G. Weber, M. Yanovsky

Numerous 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 rhythms, so understanding how the circadian clock operates within the cell will have significance 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 a broad impact on studies in humans. To study how circadian clocks are built inside cells, we use molecular and genetic approaches in 3 model systems: the plant Arabidopsis, the fruit fly Drosophila, and the mouse. 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. We constructed a model based on reciprocal regulation between TOC1 and 2 other clock factors.

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. We are investigating the mechanisms by which these 4 proteins interact to modulate gene expression; recently, we identified a major signaling pathway that regulates the activity of dCLOCK and dbMAL1.

We pioneered using gene chips to study global clock-regulated genes in 3 different model systems: Arabidopsis, Drosophila, and mouse. Currently, we are using a comparative genomics approach to further our understanding of how circadian clocks can control an incredible diversity of physiologic phenomena spanning multiple organisms. This analysis will enable us to identify conserved elements in our 3 disparate model systems and will help us determine which factors should be targeted for future research.

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 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

Alabadi, D., Oyama, T., Yanovsky, M.J., Harmon, F.G., Mas, P., Kay, S.A. Reciprocal regulation between TOC1 and LHY/CCA1 within the Arabidopsis circadian clock. Science 293:880, 2001.

Alabadi, D., Yanovsky, M.J., Mas, P., Harmer, S.L., Kay, S.A. Critical role for CCA1 and LHY in maintaining circadian rhythmicity in Arabidopsis. Curr. Biol. 12:757, 2002.

Ceriani, M.F., Hogenesch, J.B., Yanovsky, M., Panda, S., Straume, M., Kay, S.A. Genome-wide expression analysis in Drosophila reveals genes controlling circadian behavior. J. Neurosci., in press.

Harmer, S.L., Panda, S., Kay, S.A. Molecular bases of circadian rhythms. Annu. Rev. Cell Dev. Biol. 17:215, 2001.

Hogenesch, J.B., Ching, K.A., Batalov, S., Su, A.I., Walker, J.R., Zhou, Y., Kay, S.A., Schultz, P.G., Cooke, M.P. A comparison of the Celera and Ensembl predicted gene sets reveals little overlap in novel genes. Cell 106:413, 2001.

Ledger, S., Strayer, C., Ashton, F., Kay, S.A., Putterill, J. Analysis of the function of two circadian-regulated CONSTANS-LIKE genes. Plant J. 26:15, 2001.

Panda, S., Antoch, M.P., Miller, B.H., Su, A.I., Schook, A.B., Straume, M., Schultz, P.G., Kay, S.A., Takahashi, J.S., Hogenesch, J.B. Coordinated transcription of key pathways in the mouse by the circadian clock. Cell 109:307, 2002.

Schultz, T.F., Kiyosue, T., Yanovsky, M., Wada, M., Kay, S.A. A role for LKP2 in the circadian clock of Arabidopsis. Plant Cell 13:2659, 2001.

Wang, G.K., Ousley, A., Darlington, T.K., Chen, D., Chen, Y., Fu, W., Hickman, L.J., Kay, S.A., Sehgal, A. Regulation of the cycling of timeless (tim) RNA. J. Neurobiol. 47:161, 2001.

Yanovsky, M.J., Kay, S.A. Signaling networks in the plant circadian system. Curr. Opin. Plant Biol. 4:429, 2001.

Young, M.W., Kay, S.A. Time zones: a comparative genetics of circadian clocks. Nat. Rev. Genet. 2:702, 2001.