Does an ancient circadian clock control transcriptional rhythms using a non-transcriptional oscillator?

We propose to unwind a newly-discovered biological clock, that is shared by all forms of Life. The human sleep-wake cycle is the most familiar 24-hour rhythm, but in fact such 'circadian rhythms' are found in almost all living organisms. The circadian clock, which drives these rhythms, shares very similar properties in all organisms. In animals, flies, fungi, plants, archaea and cyanobacteria, it continues to generate rhythms close to 24h in duration in artificially constant environments, and its rhythms are unusually stable in duration at different temperatures. Since roughly 1995, laboratories across the world have found that the clockwork mechanism of all these organisms involves networks of gene regulation. A few key "clock genes" form a timing loop by rhythmically turning off each other's expression. Surprisingly, these overtly similar clocks depend on quite different genes in each group of organisms. The norm in biology has been that physiological processes that behave alike also share similar mechanisms, all inherited from a common ancestor. Clocks appeared to have several different origins, that gained similar behaviour through convergent evolution. This notion was reinforced when, in 2005, a non-genetic timer was discovered in cyanobacteria. The Kai oscillator rhythmically decorated a large protein with phosphate molecules, then removed them. This too seemed an idiosyncratic piece of evolution. The gene-circuit clocks in other organisms often included some control by protein phosphorylation, but their genomes lacked the Kai components that were required in cyanobacteria.

Our recent results suggest that this paradigm is wrong on two counts. At least part of the clock mechanism in an alga and in human cells does not depend on gene regulation, and this 'non-transcriptional' part of the clock appears to be shared across all organisms. Its detailed mechanism is unknown, and we propose to study it in this project. Firstly, we will follow up leads that we have recently uncovered by testing the effects of specific drugs in the alga, because the drugs have known effects on the cell's biochemistry. Secondly, we will use a technological method that we recently implemented to monitor hundreds of protein phosphorylation events in parallel, in order to find any that still remain rhythmic when gene regulation is blocked. These will represent either parts of the non-transcriptional clock, or other proteins that it controls (like the 'hands' of a mechanical clock). This part of the work will be faster and easier in the simple alga, because it has fewer protein types, and because we have found ways to study each part of the clock separately.

We will be looking back about 3 billion years in evolution, to find this earliest clock mechanism. We will ask which processes its rhythms still control today. We hope to find out why these were so important that the non-transcriptional clock has been preserved to the present. It is also important to find out how the non-transcriptional clock contributes to timing the rhythms that researchers have studied up to now, like the gene rhythms and the sleep-wake cycle. Until we know what drives the non-transcriptional clock, it will be difficult to do so, but this project should provide the tools we need. Of course, we will then test whether our results in the alga also hold in other organisms, to show whether this ancient clock still times the lives of cells in all of Biology. If so, then this original cellular timer could hold the key to future treatments for sleep disorders, to helping other algae produce biofuel while the sun shines, and to future crops that flower at predictable times in an unpredictable climate.

The 24-hour biological clock controls 15-30% of gene expression in eukaryotes. Because it regulates key signalling and metabolic pathways, this circadian system has profound effects on human health, quality of life and crop plant phenology. CSBE's clock project (Nature, 2011) showed that circadian timing in eukaryotes does not require transcriptional circuits, contrary to the 15-year-old paradigm. Unpublished results have found a key marker of the non-transcriptional clock from Archaea to humans: this clock is ancestral to the diverse, transcriptional clock circuits that are studied in eukaryotes. The functions and mechanisms of this original, cellular timer are unknown.

We aim to test the non-transcriptional clock mechanism and identify its targets, building upon our international leadership. CSBE has established a unique experimental platform in the micro-alga O.tauri, which allows us to test both the canonical, transcriptional clock and the ancient, non-transcriptional clock in the same cell type. Our protocol for transient chemical inhibition quickly gives time-resolved functional data. The small genome greatly facilitates the identification of new regulatory connections. Our preliminary data indicate that these will be representative of the green (plant) lineage.

We previously implicated three conserved protein kinases in the algal circadian system, using a chemical biology approach. We will confirm and extend the focussed studies on these candidate regulators. In parallel, a broad survey will test for rhythmic protein modification by the non-transcriptional clock. This will identify the first candidate components of this oscillator and its protein targets, and locate them within the cellular regulatory network. We will test how the ancient, non-transcriptional clock controls normal circadian rhythms, thus linking biochemical to genetic regulation. Given the apparent ubiquity of the non-transcriptional clock, our results will likely apply across Biology.

We will study a newly-discovered biological clock, which is likely to control core cellular processes upon which all Life depends. The circadian clock, the algal system, our early leads, and the public response already highlight areas of Impact.

A. Our work will benefit the ag-biotech industries by:
1. Accelerating plant biotechnology for phenology and stress traits in crops, and 3rd generation biofuels in algae, by identifying targets for manipulation in the circadian clock.
The circadian clock massively alters the plant's response to biotic and abiotic stresses, which will alter under climate change and form a key challenge to Food Security. The 'canonical' clock genes in higher plants and our alga, O.tauri, are known to control key crop traits: seasonal flowering by photoperiodism, cold acclimation, pathogen responses, and hybrid vigour. Mendel Biotechnology Inc. collaborated with us to model a relevant, clock-related pathway (now published), demonstrating the industrial relevance. This project will uncover new targets for breeding to control these pathways, in the conserved clock mechanism. CK2 is a rice flowering time gene, Hd6, for example: we will study its mode of action in the clock.
The clock modulates primary carbon metabolism (studied in our TiMet project) making it a possible target for yield enhancement in biomass and biofuel projects. O. tauri is related to hydrocarbon-producing algae. We recently modelled rhythmic starch metabolism in O. tauri, indicating that our Systems approach can facilitate metabolic engineering.

2. Enabling new approaches in agrochemical screening and plant biotech using the unicellular alga O.tauri, to reduce costs and limit environmental damage.
We showed automated, 96-well format screening of chemicals that alter dynamic gene expression in O.tauri, and also tested lethality: this is ideal for early-stage screens of herbicides (replacing Roundup), or compounds that target conserved plant signalling. High throughput will allow combinatorial screens, seeking synergistic effects from low doses of compounds with prior regulatory approval and low environmental effects, analogous to combination drug therapies. Proteomics and network analysis from this project will provide background information and protocols required to understand their mode of action. O.tauri will also facilitate genetic Mode of Action studies, for the many plant gene families that are represented in the alga by just one gene.

B. Our work will benefit the pharmaceuticals industry by suggesting new targets for treatments of ageing, parasitic and neurodegenerative disorders.
We will study conserved protein kinases with clinical indications (GSK3, insulin; CK2, cancer; CK1, chronotherapy). ~10% of O. tauri genes even have closer animal than plant homologues. Our kinase network will be relevant across the eukaryotes.
The new clock mechanism controls the peroxiredoxin (PRX) proteins across the eukaryotes. PRX is an NIH-recognised drug target in schistosomiasis. PRX is part of the antioxidant defence against ageing and abiotic stress, specifically controlled by the NMDA receptor. PRX also modulates three kinase pathways in cancer cells. Treatments targeting PRX may thus be more effective at some times of day, by mechanisms we will unravel.

C. Our work engages the public, allowing us to disseminate information on Quality of Life.
We attracted live interviews on BBC Radio 4 Today, 4 local and 5 international radio and TV channels, print coverage in over 3 national and 6 international newspapers, and an editor's post on the Scientific American blog, for our and Reddy's Nature papers in 2011. My partners in the EUCLOCK project have shown how our low-light, 24-hour society alters sleep-wake cycles and health indicators. Press coverage allows us to raise awareness of the clock's impact on health and life quality, and promote simple, research-based guidelines for living well with one's own biological clock.