Light entrainment of the circadian clock: identifying natural molecular adaptations

生物钟是一种分子起搏器，在生理、代谢、行为和其他过程中驱动日常节律，存在于从蓝藻到人类的各种生物体中。当脱离环境的线索，昼夜节律的周期（'自由运行'）略有不同，从24小时。这种内源性的节奏调整到24小时太阳日的夹带各种刺激，主要是光。现代生活引入的情况下，如跨大西洋的航班和轮班工作，昼夜节律和外部光周期是太不一样的调和;越来越多的证据表明，反复暴露于这种干扰的人遭受广泛的健康问题，包括“时差”，睡眠障碍，季节性抑郁症和癌症。在过去的几十年里，人们对生物钟的分子细节有了大量的了解。果蝇在鉴定生物钟基因方面发挥了重要作用，这些基因在哺乳动物中的序列和功能都很保守。通过诱导突变和筛选果蝇突变体，显示异常的光反应，两个蛋白质被确定为参与光转导：TIMELESS（TIM），这是一个昼夜光敏感的核心时钟蛋白，和光色素（CRY），一个专用的蓝光感光器的昼夜节律系统。这两种蛋白质相互作用，光激活的CRY附着在TIM上，迅速降解它。我们在莱斯特大学的研究重点是与昼夜光反应有关的自然遗传变异。我们的目标不是诱导随机突变，而是通过识别在不同野生种群中进化的天然变体或天然时钟等位基因来了解时钟机制，这些变体或等位基因在不同的光照和温度条件下作为分子适应。在合作与CPK和ER在莱斯特和科斯塔实验室在帕多瓦，我们已经确定了一个自然的多态性在永恒的，涉及一个单碱基插入/删除，位于两个替代翻译开始之间。我们已经发现，这种多态性遵循一个强大的纬度渐变，并保持定向选择。我们随后测试了自然分离物和转化体果蝇，发现具有不同等位基因的果蝇之间的光响应性显著不同。这种差异与我们观察到的不同等位基因的果蝇光周期反应的变化相关，这表明这种多态性代表了对寒冷环境的分子适应。最近，另一种名为JETLAG（JET）的蛋白质已被鉴定为参与TIM的光诱导降解。有趣的是，jet突变体的表型仅在携带特定的天然tim等位基因的菌株中表达。这些发现证明了自然遗传变异如何调节生物钟的光敏感性，以及如何反过来，更好地表征自然适应性，从而更好地理解生物钟机制。目前的建议旨在通过测试来自野生种群的菌株，利用定量遗传学与分子技术相结合的工具，确定生物钟基因的自然变异。我们建议使用各种全基因组筛选来识别这些变异，包括数量性状位点（QTL）定位，人工选择和全球表达分析。我们的初步QTL筛选表明，四个基因组区域（QTL），表现出显着的贡献昼夜光敏感性的变化。通过使用各种缺陷和突变株，我们将进行互补试验，使我们能够确定导致这些变异的因果序列变异。本研究的结果将使我们更好地理解光对生物钟的诱导作用，并为哺乳动物包括人类的研究提供候选基因。

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.