Functional & biochemical characterisation of circadian timekeeping mechanisms in mammalian cells

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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.