Light entrainment of the circadian clock: identifying natural molecular adaptations

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.

The current proposal is aimed to identify natural genetic variations that affect circadian light sensitivity. These variations have been driven by natural selection, serving as molecular adaptations under various light and temperature conditions. In the lab, we use light-pulse experiments to measure circadian light sensitivity: brief light stimulus is presented to animals maintained in continuous darkness. Most organisms, including Drosophila, will respond by shifting their free-running rhythm to a new phase. The size of phase shift corresponds to the light-sensitivity of the fly. We have used quantitative trait loci (QTL) mapping and identified four significant QTLs that contribute to variation in light sensitivity. To narrow down these QTL intervals, we will carry complementation tests using a series of overlapping deficiency fly strains. Crossing these deficiency strains to the QTL parental lines and testing their light response will allow us to further reduce these QTL intervals. After reducing the QTLs to several kb long, fragments will be cloned and sequenced and nucleotide variations between the QTL parental lines will be identified. Candidate alleles for different variations will be transformed into flies by homologous recombination. Comparing the light sensitivity of these transgenic flies that express different natural alleles will indicate which polymorphic sites are important for circadian light sensitivity. We will also use artificial selection to produce lines that differ in their light sensitivity. The response to selection will provide information about the genetic architecture of this trait, and the selected lines will be tested for divergent gene expression. Differentially expressed genes will be sequenced and polymorphic sites will be identified and tested as above. Finally, we will also exploit variation in light sensitivity between two Drosophila species using custom microarrays and transformant flies.