Functional & biochemical characterisation of circadian timekeeping mechanisms in mammalian cells

日常节律是陆地生物的生物化学、生理和行为的基础。在人类中，24小时的周期与睡眠和觉醒模式、血液皮质醇水平、认知功能以及每个细胞中约20%的基因组表达有着密不可分的联系。这些节律失调与代谢性疾病和癌症易感性增加有关。此外，时间药理学的新兴领域旨在利用生理和代谢的日常变化，以最大限度地提高药物的治疗效果。细胞水平的昼夜节律是由转录、翻译和翻译后因子网络调控的。人们普遍认为，细胞时钟是由一个核心的转录反馈机制驱动的；主要元件是激活转录因子CLOCK和BMAL1，以及它们的抑制因子：Period （PER）和Cryptochrome （CRY）。CRY被认为是主要的抑制因子，尽管其全部功能需要PER，因此CRY突变体已被用作心律失常的动物和细胞模型。然而，O’neill实验室在PER2::LUC生物发光报道的CRY1和CRY2纯合子缺失的小鼠细胞中观察到持续的，尽管不那么强劲的昼夜节律。我们发现这些与cry无关的PER2::LUC节律受到翻译后机制的调节，例如酪蛋白激酶的磷酸化，这也有助于野生型细胞的计时。本项目将以这些先前的发现为基础，通过药理学操作和串联质谱法，确定野生型和哭泣细胞中维持计时的关键分子成分。此外，它将利用尖端的遗传操作工具（如诱导表达和CRISPR-Cas9基因组编辑）来表征这些途径的功能，以确定维持细胞昼夜节律所必需和足够的最小时钟组件集。这一知识将揭示代谢疾病和癌症的新治疗靶点，并为时间药理学开辟新的途径。

Daily rhythms underlie the biochemistry, physiology and behavior of terrestrial life. In humans, the 24-hour cycle is inextricably linked to patterns of sleep and wake, blood cortisol levels, cognitive function and the expression of around 20% of the genome in each cell. Dysregulation of these rhythms is associated with metabolic diseases and increased cancer susceptibility. In addition, the emerging field of chronopharmacology aims to take advantage of the daily changes of physiology and metabolism in order to maximize the therapeutic effect of drugs.Circadian rhythms at the cellular level are regulated by networks of transcriptional, translational and post-translational factors. It is widely accepted that the cellular clockwork is driven by a core transcriptional feedback mechanism; the main elements being activating transcription factors CLOCK and BMAL1, and their repressors: Period (PER) and Cryptochrome (CRY). CRY is regarded as the main repressor, although PER is required for its full function, and so CRY mutants have been used as arrhythmic animal and cellular models. However, the O'Neill lab have observed persistent, albeit less robust, circadian rhythms in cells from mice that are homozygous null for CRY1 and CRY2 - reported by PER2::LUC bioluminescence. We find that these CRY-independent PER2::LUC rhythms are regulated by post-translational mechanisms, such as phosphorylation by casein kinase , that also contribute to timekeeping in wild type cells. This project will build upon these prior findings by identifying the key molecular components that sustain timekeeping in both wild type and CRY-deficient cells, though pharmacological manipulation and tandem mass spectrometry. Furthermore it will aim to characterize the function of these pathways using cutting edge tools for genetic manipulation such as inducible expression and CRISPR-Cas9 genome editing, in order to determine the minimal set of clock components that are necessary and sufficient to sustain cellular circadian rhythms. This knowledge will reveal novel therapeutic targets for metabolic diseases and cancers, as well as opening new avenues for chronopharmacology.