A Luciferase-Based Assay to Test Whether Gene Expression Responses to Environmental Inputs Are Temporally Restricted by the Circadian Clock
Gating is the mechanism by which the influence of an environmental signal on a particular output is temporally restricted by the circadian clock, so that the maximum response of the output to the signal occurs at a specific time. Gated regulation mechanisms have been described for several genes whose expression is strongly induced by light or temperature at certain times but repressed by the circadian clock at others. To reveal a gated pattern of expression in response to light, light pulses are applied in the dark at different times of the 24 h cycle and the transcriptional response of the gene of interest is then monitored with an appropriate technique. Luciferase (LUC) reporters have been the method of choice to study circadian rhythms in the past decades, but this methodology also provides an ideal platform for performing a gating assay. In this chapter, we describe a LUC imaging based protocol designed to test whether the influence of light on the expression of a gene of interest is gated by the circadian clock.
Key wordsCircadian clock Gating Gene expression Light Luciferase
The laboratory of George Coupland receives a core grant from the Max Planck Society. We are grateful to Reka Tóth and Seth Davis for many discussions on luminescence measurements and experimental design.
- 10.Edwards KD, Akman OE, Knox K, Lumsden PJ, Thomson AW, Brown PE, Pokhilko A, Kozma-Bognar L, Nagy F, Rand DA, Millar AJ (2010) Quantitative analysis of regulatory flexibility under changing environmental conditions. Mol Syst Biol 6:424Google Scholar