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On the Robustness of the Mammalian Circadian Oscillator: Dissection of Molecular Mechanisms

C. Dibner, D. Sage, M. Unser, T. d'Eysmond, F. Naef, Ü. Schibler

The 2009 Gordon Conference on Chronobiology: Molecular Mechanisms of Circadian Clocks (GRC'09), Newport RI, USA, July 19-24, 2009.



The mammalian circadian clock is organized in a hierarchical manner in that a master pacemaker residing in the suprachiasmatic nucleus (SCN) synchronizes slave oscillators existing in most body cells. Central and peripheral clocks have a similar molecular makeup, and both are thought to rely on transcription feedback loops in clock gene expression. The major and essential loop involves the autorepression of cryptochrome (Cry1, Cry2) and period (Per1, Per2) genes. This rhythm generating circuitry is functional in most cell types, including primary or immortalized cultured fibroblasts, which harbor self-sustained and cell-autonomous oscillators resilient to temperature changes and cell division. We examined the robustness of circadian oscillators by subjecting cultured cells to large fluctuations of temperature and global transcription rates. Our studies revealed that an up to three-fold reduction in RNA polymerase II (Pol II) dependent transcription did not abolish circadian oscillations, although it did dampen the magnitudes and amplitudes of cyclic gene expression. Remarkably, the attenuation of transcription rates accelerated the clocks of both NIH3T3 cells and mouse tail tip fibroblasts. In contrast, inhibition of PolII dependent transcription by either actinomycin D or α-amanitin had no effect on the period length of primary fibroblasts from Per1KO mice. This suggests a possible role for Per1 in transcription compensation of circadian pacemakers. Single cell bioluminescence analysis by time lapse microscopy further confirmed that upon reduced Pol II transcription, individual fibroblast clocks were less well synchronized and exhibited shorter oscillation periods and lower magnitudes, which explains the generally lower magnitude at the population level of these cells. To analyze the large data sets obtained by time lapse microscopy, we have developed Circadian Gene Express (CGE)—a novel software compatible with the ImageJ package. The ability of circadian clocks to keep running accurately despite significant variation in transcription rates may be necessary for the oscillator to maintain synchrony under different physiological conditions.


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