Light entrainment of the circadian clock: identifying natural molecular adaptations

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Circadian clocks are molecular pacemakers that drive daily rhythms in physiology, metabolism, behaviour and other process, and are present in diverse range of organism, from cyanobacteria to human. When detached from ambient cues, circadian clock cycle ('freerun') at periods slightly different from 24 hr. This endogenous rhythm is adjusted to the 24 hr solar day by entrainment to various stimuli, primarily light. Modern life introduces situations, such as trans-atlantic flights and shift work, where the circadian rhythm and the external light cycle are too dissimilar to reconcile; accumulating evidence suggests that people exposed repeatedly to such disruptions suffer from wide range of health problems, including 'jetlag', sleep disorders, seasonal depression, and cancer. In the last few decades, a great deal has been learned about the molecular details of the clock. Drosophila has been instrumental in identifying circadian clock genes, which are well conserved in mammals, both in sequence and function. By inducing mutagenesis and screening for Drosophila mutants that show aberrant light response, two proteins were identified to be involved in light transduction: TIMELESS (TIM), which is a circadian light-sensitive core-clock protein, and CRYPTOCHROME (CRY), a dedicated blue-light photoreceptor of the circadian system. These two proteins interact with each other, and light-activated CRY attaches itself to TIM, degrading it rapidly. Our research at the University of Leicester focuses on natural genetic variation related to circadian photo-responsiveness. Rather than inducing random mutations, we aim to understand the clock mechanism by identifying natural variants, or natural clock alleles that have been evolved in different wild populations, serving as molecular adaptations under different light and temperature conditions. In collaboration with CPK and ER at Leicester and the Costa lab at Padova, we have identified a natural polymorphism in Timeless that involves a single-base insertion/deletion, situated between two alternative translation starts. We have found that this polymorphism follows a robust latitudinal cline and is maintained by directional selection. We subsequently tested natural isolates and transformants flies and found that photo-responsiveness is significantly different between flies with the different alleles. This difference was correlated with the variation we have observed in the photoperiodic response of flies with the different alleles suggesting that this polymorphism represents molecular adaptation to cold environments. Recently, another protein named JETLAG (JET) has been identified as being involved in light-induced degradation of TIM. Interestingly, it turns out that the phenotype of jet mutants is only expressed in strains carrying a specific natural tim allele. These discoveries have demonstrated how natural genetic variation modulates light sensitivity of the circadian clock, and how in turn, could the better characterization of natural adaptations lead to a better understanding of the circadian-clock mechanism. The current proposal is aimed at identifying natural variation in clock genes by testing strains derived from wild-populations, using tools of quantitative genetics combined with molecular techniques. We propose to use various genome-wide screens to identify these variations, including Quantitative Trait Loci (QTL) mapping, artificial selection and global expression analysis. Our preliminary QTL screen indicated four genomic regions (QTLs) that show significant contribution to variation in circadian light-sensitivity. By using various deficiency and mutant strains we will carry complementation tests that will allow us to identify the causal sequence variations that account for these variations. The results of this study will allow a better understanding of light entrainment of the clock, and provide candidate genes for studying in mammals, including human.