Light entrainment of the circadian clock: identifying natural molecular adaptations

生物钟是驱动生理、代谢、行为和其他过程的日常节律的分子起搏器，存在于从蓝藻到人类的各种生物中。当脱离环境信号时，生物钟周期（“自由运行”）的周期与24小时略有不同。这种内源性节律通过各种刺激（主要是光）调整为24小时太阳日。现代生活引入了一些情况，例如跨大西洋航班和轮班工作，昼夜节律和外部光周期太不相似而无法调和；越来越多的证据表明，经常受到这种干扰的人会出现各种各样的健康问题，包括“时差反应”、睡眠障碍、季节性抑郁症和癌症。在过去的几十年里，人们对时钟的分子细节已经了解了很多。果蝇在识别生物钟基因方面发挥了重要作用，这些基因在哺乳动物中无论是在序列上还是在功能上都很保守。通过诱导诱变和筛选显示异常光反应的果蝇突变体，确定了两种参与光转导的蛋白：TIMELESS (TIM)，这是一种昼夜节律光敏核心时钟蛋白，以及CRYPTOCHROME (CRY)，一种昼夜节律系统专用的蓝光光感受器。这两种蛋白相互作用，光激活的CRY附着在TIM上，迅速降解TIM。我们在莱斯特大学的研究重点是与昼夜节律光反应相关的自然遗传变异。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自然变异或自然时钟等位基因来了解时钟机制，这些等位基因在不同的光和温度条件下起着分子适应作用。在与莱斯特的CPK和ER以及帕多瓦的Costa实验室的合作中，我们已经确定了Timeless的自然多态性，该多态性涉及位于两个替代翻译起始点之间的单碱基插入/删除。我们发现这种多态性遵循一个强大的纬度梯度，并通过方向选择维持。随后，我们对天然分离蝇和转化蝇进行了测试，发现不同等位基因的蝇对光的反应有显著差异。这种差异与我们在不同等位基因的果蝇光周期反应中观察到的差异有关，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）的蛋白质被确定参与光诱导的TIM降解。有趣的是，事实证明喷气突变体的表型仅在携带特定天然时间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，以及反过来如何更好地表征自然适应，从而更好地理解生物钟机制。目前的建议旨在通过使用定量遗传学和分子技术相结合的工具，通过测试来自野生种群的菌株来确定时钟基因的自然变异。我们建议使用各种全基因组筛选来识别这些变异，包括定量性状位点（QTL）定位、人工选择和全局表达分析。我们的初步QTL筛选显示，四个基因组区域（QTL）对昼夜节律光敏感性的变化有重要贡献。通过使用各种缺陷和突变菌株，我们将进行互补测试，这将使我们能够确定导致这些变化的因果序列变化。这项研究的结果将使人们更好地理解生物钟的光携带，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.