Potential contribution of tandem circadian enhancers to nonlinear oscillations in clock gene expression.

Limit-cycle oscillations require the presence of nonlinear processes. Although mathematical studies have long suggested that multiple nonlinear processes are required for autonomous circadian oscillation in clock gene expression, the underlying mechanism remains controversial. Here we show experimentally that cell-autonomous circadian transcription of a mammalian clock gene requires a functionally interdependent tandem E-box motif; the lack of either of the two E-boxes results in arrhythmic transcription. Although previous studies indicated the role of the tandem motifs in increasing circadian amplitude, enhancing amplitude does not explain the mechanism for limit-cycle oscillations in transcription. In this study, mathematical analysis suggests that the interdependent behavior of enhancer elements including not only E-boxes but also ROR response elements might contribute to limit-cycle oscillations by increasing transcriptional nonlinearity. As expected, introduction of the interdependence of circadian enhancer elements into mathematical models resulted in autonomous transcriptional oscillation with low Hill coefficients. Together these findings suggest that interdependent tandem enhancer motifs on multiple clock genes might cooperatively enhance nonlinearity in the whole circadian feedback system, which would lead to limit-cycle oscillations in clock gene expression.