Characterization of a novel circadian regulating gene crtc in Drosophila melanogaster

Abstract

Most living organisms have a biological clock and it has been highly conserved from bacteria to mammals. Much of our current understanding of circadian clock system in mammals is driven from many studies done in Drosophila. Especially, Drosophila melanogaster is a good model system for clarifying the molecular mechanism underlying the circadian rhythms because this model organism is easy to perform the genetic manipulation and large-scale mutant screening. In addition, mammals and Drosophila share the similar fundamental clock mechanism. Endogenous circadian clocks have a period of approximately 24 hours. They not only persist their circadian rhythms in constant conditions but also can be easily entrained by environmental signals such as light, temperature and other external stimuli. Light is one of the strongest environmental time cues for entraining endogenous circadian rhythms. Emerging evidence indicates that CREB-regulated transcription co-activator 1 (CRTC1) is a key player in this pathway, stimulating light-induced Period1 (Per1) transcription in mammalian clocks. Here, we demonstrate a light-independent role of Drosophila CRTC in sustaining circadian behaviors. Genomic deletion of the crtc locus causes long but poor locomotor rhythms in constant darkness. Overexpression or RNA interference-mediated depletion of CRTC in circadian pacemaker neurons similarly impairs the free-running behavioral rhythms, implying that Drosophila clocks are sensitive to the dosage of CRTC. The crtc null mutation delays the overall phase of circadian gene expression yet it remarkably dampens light-independent oscillations of TIMELESS (TIM) proteins in the clock neurons. In fact, CRTC overexpression enhances CLOCK/CYCLE (CLK/CYC)-activated transcription from tim but not per promoter in clock-less S2 cells whereas CRTC depletion suppresses it. Consistently, TIM overexpression partially but significantly rescues the behavioral rhythms in crtc mutants. Taken together, our data suggest that CRTC is a novel co-activator for the CLK/CYC-activated tim transcription to coordinate molecular rhythms with circadian behaviors over a 24-hour time-scale. We thus propose that CRTC-dependent clock mechanisms have co-evolved with selective clock genes among different species