News and Publications

Molecular Genetics of Circadian Clocks and PAS Domain Signaling Proteins

S.A. Kay, C. Andersson, F. Ceriani, T. Darlington, P. Devlin, J. Kreps, T. Kuhlmann, S. Panda, R. Raman, T. Schultz, D. Somers, C. Strayer, K. Wager-Smith, A. Scully, S. Harmer

Almost all cellular processes fluctuate with a 24-hour periodicity, and these periodicities are known as circadian rhythms. The circadian biological clock controls diverse events, from the sleep-wake cycle in humans to the overall rate of photosynthesis in plants. Many pathologic changes in humans, such as sleep disorders, are most likely associated with a defect in circadian rhythm, so understanding how cells generate these 24-hour rhythms 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 for studies in humans. To study how circadian clocks are built inside of cells, we are using molecular and genetic approaches in 2 model systems: the plant Arabidopsis and the fruit fly Drosophila.

In Arabidopsis, we have detected several genes that have circadian-regulated transcription. One of these, CAB2, encodes a protein essential for photosynthesis; transcription of the gene 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 gene for luciferase. Transgenic plants containing this construct are imaged by using highly sensitive video cameras. This enables us to measure transcription noninvasively in living tissues and cells, where rhythmic bioluminescence reflects clock control of gene expression. We are using this real-time imaging assay to determine the specific transcription factors and signaling pathways involved in clock-regulated transcription.

In a complementary approach, we screened mutants to look for seedlings that "glow" with an altered rhythm. We detected mutants with aberrant circadian function, and we are cloning several of these loci by using chromosome walking. We expect that these genes will encode clock components, the molecular cogs that drive circadian rhythms.

In Drosophila, we are interested in elucidating how circadian clocks are organized to control behavior and physiology in animals. Two circadian clock genes have been identified to date: period (per) and timeless (tim). These genes form part of an autoregulatory feedback loop of transcription, whereby the proteins PER and TIM repress transcription of their own genes. To elucidate the function of these genes further, we generated transgenic Drosophila that express both per and the gene for luciferase. We have developed an automated assay in which live flies are placed in the wells of microtiter plates, where they ingest luciferin in their food substrate. The insects then begin to emit bioluminescence from per-expressing tissues, which we monitor by using robotic plate-reading luminometers.

Thus, we are now able to measure transcription in real time in living animals. This technique has enabled us to determine which tissues express per and the exact kinetics of cyclic per transcription in whole animals. Using green fluorescent protein as a parallel reporter in confocal microscopy, we developed culture techniques for these tissues. We discovered that contrary to the accepted dogma, independent circadian clocks exist in several tissues outside the brain, and we are determining the properties of these cellular rhythms.

We are using this automated bioluminescence system and classical and enhancer-trap mutant screens to isolate additional Drosophila clock mutants. We recently cloned 2 new clock genes from Drosophila, dclock and dbMAL1, and showed that these genes play a central role in the clock mechanism. These proteins are both members of the bHLH-PAS transcription factor family. We are attempting to determine the structure of the PAS domain in collaboration with colleagues here at TSRI.

We also developed the use of green fluorescent protein and blue fluorescent protein to measure the dimerization of transcription factors in live cells, by measuring fluorescence resonance energy transfer. Ultimately, we wish to measure spatiotemporal control of "protein sociology" and gene transcription kinetics in living cells.

PUBLICATIONS

Andersson, C.R., Kay, S.A. COP1 and HY5 interact to mediate light-induced gene expression. Bioessays 20:445, 1998.

Darlington, T., Wager-Smith, K., Ceriani, F., Staknis, D., Gekakis, N., Steeves, T., Weitz, C., Takahashi, J., Kay, S.A. Closing the circadian loop: CLOCK-induced transcription of its own inhibitors per and tim. Science 280:1599, 1998.

Genoud, T., Millar, A.J., Nishizawa, N., Kay, S.A., Schafer, E., Nagatani, A., Chua, N.-HAn Arabidopsis mutant hypersensitive to red and far-red light signals. Plant Cell 10:889, 1998.

Guesz, M., Fletcher, C., Block, G., Straume, M., Copeland, N.G., Jenkins, N.A., Kay, S.A., Day, R. Long-term monitoring of circadian rhythms in c-fos gene expression from suprachiasmatic nucleus cultures. Curr. Biol. 7:758, 1997.

Kolar, C., Fejes, É., Ádám, E., Schäfer, S., Kay, S.A., Nagy, F. Transcription of Arabidopsis and wheat CAB genes in single tobacco transgenic seedlings exhibits independent rhythms in a developmentally regulated fashion. Plant J. 13:563, 1998.

Kreps, J., Kay, S.A. Coordination of metabolism and development by the circadian clock. Plant Cell 9:1235, 1997.

Pellequer, J.-L., Wager-Smith, K., Kay, S.A., Getzoff, E. Photoactive yellow protein: A structural prototype for the three-dimensional fold of the PAS domain superfamily. Proc. Natl. Acad. Sci. U.S.A. 95:5884, 1998.

Plautz, J.D., Kay, S.A. Synchronous real-time reporting of multiple cellular events. Methods Cell Biol. 58:283, 1998.

Somers, D., Webb, A., Pearson, M., Kay, S.A. The short period mutant, toc1-1, alters circadian clock regulation of multiple outputs throughout development in Arabidopsis thaliana. Development 125:485, 1998.

Stanewsky, R., Jamison, C., Plautz, J., Kay, S.A., Hall, J.C. Two circadian-regulated elements contribute to cycling period gene expression in Drosophila. EMBO J. 16:5006, 1997.

Welsh, S., Kay, S.A. Reporter gene expression for monitoring gene transfer. Curr. Opin. Biotechnol. 8:617, 1997.