Kevin A. Feeney1, Louise L. Hansen2, Marrit Putker1, Consuelo Olivares-Yañez3, Jason Day4, Lorna J. Eades5, Luis F. Larrondo3, Nathaniel P. Hoyle1, John S. O'Neill1,#, and Gerben van Ooijen2,#

1MRC Laboratory for Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH, UK2School of Biological Sciences, University of Edinburgh, Max Born Crescent, Edinburgh EH9 3BF, UK3Millennium Nucleus for Fungal Integrative and Synthetic Biology, Departamento de Genética Molecular y Microbiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Casilla 114-D, Santiago, Chile4Department of Earth Sciences, University of Cambridge, Downing St, Cambridge CB2 3EQ, UK5School of Chemistry, University of Edinburgh, David Brewster Road, Edinburgh EH9 3FJ, UK

Abstract
Circadian clocks are fundamental to the biology of most eukaryotes, coordinating behavior and physiology to resonate with the environmental cycle of day and night through complex networks of clock-controlled genes1–3. A fundamental knowledge gap exists however, between circadian gene expression cycles and the biochemical mechanisms that ultimately facilitate circadian regulation of cell biology4,5. Here we report circadian rhythms in the intracellular concentration of magnesium ions, [Mg2+]i, which act as a cell-autonomous timekeeping component to determine key clock properties in both a human cell line and a unicellular alga that diverged from metazoans more than 1 billion years ago6. Given the essential role of Mg2+ as a cofactor for ATP, a functional consequence of [Mg2+]i oscillations is dynamic regulation of cellular energy expenditure over the daily cycle. Mechanistically, we find that these rhythms provide bilateral feedback linking rhythmic metabolism to clock-controlled gene expression. The global regulation of nucleotide triphosphate turnover by intracellular Mg2+ availability has potential to impact upon many of the cell’s >600 MgATP-dependent enzymes7 and every cellular system where MgNTP hydrolysis becomes rate limiting. Indeed, we find that circadian control of translation by mTOR8 is regulated through [Mg2+]i oscillations. It will now be important to identify which additional biological processes are subject to this form of regulation in tissues of multicellular organisms such as plants and humans, in the context of health and disease.

Circadian rhythms occur cell-autonomously and are not restricted to metazoans or multicellular organisms, being found throughout eukaryotes and some prokaryotes9. Although explicit clock gene identities share no similarity across phylogenetic kingdoms, in every case temporal orchestration of gene expression is driven by timekeeping mechanisms that result in rhythmic clock protein transcription factor activity. In human cells, for example, a heterodimeric complex of BMAL1 and CLOCK positively regulates the expression of genes (Period1/2, Cryptochrome1/2) that encode its own repressor complex9, whereas in the marine unicellular alga Ostreococcus tauri, a reduced version of the stereotypical plant-like circadian clock consists of a feedback loop between the morning-expressed MYB-like transcription factor CCA1 and the evening-expressed protein TOC110. Intriguingly, the role of enzymes such as casein kinase 1 and 2 in the post-translational regulation of clock protein activity/stability, and the speed at which biological clocks run, is functionally conserved across eukaryotes11. Also, we recently reported a circadian rhythm in the redox state of peroxiredoxin proteins that is conserved across phylogenetic kingdoms4,12. Critically, this metabolic rhythm persists in the absence of nascent gene expression, both in human cells (anucleate erythrocytes)13 and in Ostreococcus, which ceases mRNA production upon prolonged photosynthetic inactivity under constant darkness14, indicating that circadian regulation of cellular metabolism is not strictly reliant upon rhythmic transcription. These and a number of other observations render it plausible that circadian rhythms observed in diverse eukaryotes incorporate features of a post-translational timing mechanism that was present in the last eukaryotic common ancestor, LECA15.
A rich diversity of evolutionarily conserved membrane transporters and channels mediate uptake of ions and micronutrients essential for cellular biochemistry, with several studies having reported their circadian regulation in a variety of contexts (e.g.16), and with membrane models of the circadian clock actually predating the identification of any clock genes17,18. It is plausible that rhythmic regulation of ion transport may have conferred an adaptive advantage upon early eukaryotes, allowing the global regulation of biochemical equilibria and reaction rates to tune cellular metabolism with environmental cycles. We therefore asked whether circadian regulation of transmembrane ion transport might constitute a fundamental feature of circadian timekeeping in eukaryotic cells. To find evidence for such regulation in modern organisms, we compared two eukaryotic lineages separated by ~1.5 billion years of evolution6: human U2OS cells and Ostreococcus tauri.
Inductively Coupled Plasma Mass Spectrometry (ICP-MS) was employed to generate an unbiased analysis of the total cellular elemental composition, including all organelles and cellular structures19,20, over circadian time series. In Ostreococcus, clear daily rhythms were detected under natural light/dark cycles for many different ions, including potassium and magnesium (Fig. 1a and Extended Data Fig. 1). Analyses of cells maintained under constant light revealed that, whilst rhythms of some metal ions ceased under these conditions, oscillations in potassium and magnesium persisted, indicating their regulation by cell-autonomous circadian clock mechanisms. Strikingly, circadian rhythms of magnesium and potassium were also observed in non-proliferating human U2OS cells maintained over three days under constant conditions (Fig. 1b and Extended Data Fig. 2). The oscillation in intracellular potassium is likely to be a consequence of rhythmic Na+-dependent pump activity, as circadian regulation of sodium-dependent solute-transport and plasma membrane ATPases has been reported widely (e.g.2,16,21,22). Calcium is largely found in, and released from, intracellular stores and although calcium has a clearly established role in circadian rhythms11, we infer the absence of any obvious cell-autonomous Ca2+ oscillations to mean that its net cellular flux does not vary over the circadian cycle in these cells. We considered the oscillation in Mg2+ to be of particular interest, since Mg2+ is an essential cofactor for (deoxy-) nucleotide triphosphates. Cellular Mg2+ therefore has the potential to regulate many intracellular metabolic reactions, through its requirement for the activities of >600 enzymes7, including those involved in ATP production, as well as DNA, RNA and protein synthesis. Furthermore, free intracellular Mg2+ can act as a second messenger. For example, epidermal growth factor stimulation induces a rapid increase in [Mg2+]i, which acts via highly Mg-sensitive mTOR to activate protein synthesis without any change in total ATP levels23.
Firstly, we exploited the role of Mg2+ as an ATP cofactor, to measure freely available intracellular magnesium concentrations in cellular extracts using the MgATP-dependent enzyme firefly luciferase (Fig. 1c,d). Our initial ICP-MS observations were confirmed using this assay, and we observed clear rhythms of [Mg2+]i over two days under constant conditions in both cell types. Bioluminescent reporters for clock gene activity, recorded in parallel, confirmed the [Mg2+]i oscillation to occur roughly in antiphase with circadian markers normally expressed around (subjective) dawn (CCA1-LUC, Per2:luc, Fig. 1a,b). Conservation between human and algal cells indicates that rhythmic cation transport might constitute a general feature of cellular rhythmicity. We therefore investigated whether such oscillations were also present in the fungus Neurospora crassa, representing the third eukaryotic kingdom. Similar to our observations in algal and human cells, a circadian rhythm in cellular magnesium content was observed in antiphase with the abundance of the clock protein FRQ9 (Extended Data Fig. 3a-c). We also observed cellular magnesium rhythms in cultured mouse fibroblasts isolated from adult lung, indicating that magnesium rhythms are also present in non-transformed, terminally differentiated mammalian cells (Extended Data Fig. 3d). These rhythms were disrupted in fibroblasts isolated from Cryptochrome1-/-,Cryptochrome2-/- mice, suggesting their dependence upon clock gene activity. Incidentally, we note that approximately 24 h [Mg2+]i rhythms occur cell-autonomously, are temperature-compensated (Extended Data Fig. 4) and entrain to relevant external cues and are therefore circadian by definition24.
The rhythm in total cellular Mg2+ measured by ICP-MS must result from daily cycles between net cellular Mg2+ influx and efflux, through circadian regulation of plasma membrane Mg2+ channel and transporter activity7. All known magnesium transporting proteins in animals (channels TRPM7, MAGT1, MMGT1 and CNMM3, as well as Mg2+-transporter SLC41) exhibit circadian rhythms at the mRNA level in four or more tissues25 (Extended Data Fig. 5). Ostreococcus encodes homologs of TRPM7, CNMM3 and SLC41, which are also differentially expressed over the daily cycle (Extended Data Fig. 5).

Moreover, siRNA-mediated knockdown of each Mg2+-channel/transporter in U2OS cells results in lengthened circadian period26, suggesting that as well as being clock-regulated, [Mg2+]i might also feed back to regulate the cellular clock.
To determine whether [Mg2+]i oscillations are relevant to timekeeping mechanism therefore, we next employed inhibitors of magnesium transport. Cobalt(III)hexammine (Co(NH3)62+, CHA) and cobalt(III)chloro-pentammine (Co(NH3)5Cl2+, CPA) closely resemble a single-solvation shell hydrated Mg2+ ion, and have been shown to block Mg2+ transport through at least two different transporters/channels27,28. We found both compounds to dose-dependently increase [Mg2+]i in both cell types (Fig. 2a,b and Extended Data Fig. 6), indicating that these compounds do act to block Mg2+ transport. Increased [Mg2+]i was associated with clear dose-dependent lengthening of circadian period (Fig. 2c-f). Importantly, the effects of CHA on circadian period were dependent on the concentration of extracellular magnesium (Extended Data Fig. 7a-d), indicating a specific role for magnesium in determining the speed at which both algal and human cellular clocks run. To further substantiate this observation, we used quinidine, an inhibitor that acts on several ion transport activities including the SLC41 Na+/Mg2+ antiporter29. Similarly to CHA and CPA, quinidine led to dose-dependent accumulation of intracellular Mg2+ and lengthening of circadian period in both cell types (Fig. 2). SLC41 constitutes the sole protein known to exhibit sodium-dependent Mg2+-transport activity29 that is conserved between human and Ostreococcus cells and so, to test its specific cellular clock function, we performed siRNA-mediated SLC41 knock-down: observing a clear Mg2+-dependent lengthening of circadian period (Extended Data Fig. 7e-g).
We also observed that depletion of magnesium from the growth medium led to reduced [Mg2+]i, and had dramatic effects on the amplitude and period of the circadian clock in Ostreococcus (Fig. 3a,b). Although prolonged growth in low Mg2+ media had adverse effects on cell viability of the U2OS line, cells that were simply transferred to Mg2+-free media showed reduced [Mg2+]i and exhibited circadian rhythms with increased period and decreased bioluminescence amplitude relative to normal media controls (Fig. 3c,d). In neither case was the effect of [Mg2+]i-depletion attributable to decreased ATP availability, since in both cases cellular ATP levels were significantly increased (Fig. 3b,d).
Thus, as observed previously for cAMP signalling30, treatments which constitutively elevate or reduce [Mg2+]i both result in period lengthening of clock gene expression in these cell types, indicating that dynamic circadian regulation of [Mg2+]i might be a cellular clock component. On the other hand however, it remained possible that Mg2+ transport might not contribute to clock mechanism, but instead simply be permissive for cellular timekeeping, analogous to the function of ‘housekeeping’ genes. To distinguish between these two possibilities, we determined whether an enforced transition in [Mg2+]i acts as a state variable for cellular circadian oscillations. Upon introduction of magnesium to Ostreococcus cells starved of magnesium, we observed strict resetting of the subsequent rhythm to subjective dawn, regardless of prior circadian phase, indicating that changes in [Mg2+]i can act as a strong zeitgeber (Extended Data Fig. 8). Therefore, our data indicate that not only does [Mg2+]i exhibit a bona fide circadian rhythm across diverse eukaryotic cells, but also that appropriate manipulation of [Mg2+]i is sufficient to determine the key properties of the oscillation (period, amplitude, and phase), making [Mg2+]i indistinguishable from a core clock component.
We considered that the increased cellular ATP levels we observed during Mg2+ depletion might be attributable to differential sensitivity of MgATP-dependent cytosolic enzymes compared with the organellar ATP synthesis machinery. For example, ATP accumulation was accompanied by a marked reduction in extracellular lactate accumulation in U2OS cultures (Extended Data Fig. 9a), indicative of reduced glycolysis. We therefore considered whether rate-changes in gross cytosolic energy metabolism might be a functional consequence of cell-autonomous circadian [Mg2+]i oscillations. A clear prediction would be that global rates of translation should be limited at circadian phases of low [Mg2+]i, since protein synthesis is one of the most energetically expensive processes that cells undertake.
We assayed translation rate by puromycin incorporation8 in both cell types just before (anticipated) biological dusk and dawn; at the phase of lowest and highest [Mg2+]i, respectively. The Ostreococcus experiment was performed under its natural light/dark cycle so as to best model an organism in its natural environment, whereas the U2OS experiment was performed under constant conditions to model innate peripheral cellular clock function. We observed that both Ostreococcus and U2OS cells did exhibit significantly higher translation rates at the peak of [Mg2+]i, as predicted (Fig. 4c,d). In mammalian cells, the highly MgATP-sensitive mTOR pathway was recently shown to mediate circadian control of translation8. We hypothesised that [Mg2+]i oscillations might act via mTOR to effect circadian translational regulation, and tested this using two pharmacologically distinct mTOR inhibitors (torin1 and rapamycin). Both inhibitors lengthened period dose-dependently, and abolished any additional period lengthening due to depletion of extracellular magnesium that was observed in controls. This clear ‘ceiling effect’ strongly suggests changes in [Mg2+]i act through mTOR activity to regulate cellular circadian period (Extended Data Fig. 9b-e). To test the extent to which marked differences in translation rates between dawn and dusk were attributable to cell-autonomous [Mg2+]i oscillations, we incubated cells acutely with CHA to block magnesium transport before assaying overall translational rates. CHA significantly attenuated the difference in [Mg2+]i between dawn and dusk (Fig. 4a), and also phase-dependently affected ATP levels (Fig. 4b). Crucially, differential translation rates were attenuated similarly by CHA treatment (Fig. 4c,d), indicating that in both cell types, circadian regulation of magnesium levels contributes to circadian rhythms in global translation rate.
Our data support a model (Fig. 4e, Extended Data Fig. 10a) where the cellular clockwork regulates the expression of plasma membrane Mg2+ channels and transporters to generate rhythmic magnesium fluxes. These rhythms appear to facilitate the higher energetic demands and protein production of human cells during the (biological) day, as well as the global down-regulation of ATP turnover and translation in photosynthetic cells at night. Reciprocally, the [Mg2+]i rhythm feeds back to regulate the period, phase and amplitude of clock gene expression rhythms, acting as a “meta-regulator” to integrate metabolic rhythms with transcriptional feedback models of cellular timekeeping. It is noteworthy however, that the magnesium rhythm observed in Ostreococcus persists under transcriptionally inactive conditions14 of constant darkness (Extended Data Fig. 10b,c), in the absence of transcriptional regulation of membrane transport. It is therefore likely that some aspects of circadian magnesium flux are regulated by, and may contribute to, the same uncharacterised non-transcriptional clock mechanism13,14 that also drive persistent peroxiredoxin overoxidation rhythms in transcriptionally inactive cells across taxa, and which we speculate was present in the LECA.
Cell-autonomous rhythms in [Mg2+]i availability have the potential to impart circadian regulation to any cellular system where MgNTP hydrolysis becomes rate limiting. Although the clinical relevance of [Mg2+]i in various tissues is beginning to garner more attention, the interactions between magnesium transport and human health are poorly understood. Further investigation of the downstream consequences of circadian regulation of [Mg2+]i will therefore be important.