Circadian clocks in human red blood cells

Journal name:
Nature
Volume:
469,
Pages:
498–503
Date published:
DOI:
doi:10.1038/nature09702
Received
Accepted
Published online

Abstract

Circadian (~24 hour) clocks are fundamentally important for coordinated physiology in organisms as diverse as cyanobacteria and humans. All current models of the molecular circadian clockwork in eukaryotic cells are based on transcription–translation feedback loops. Non-transcriptional mechanisms in the clockwork have been difficult to study in mammalian systems. We circumvented these problems by developing novel assays using human red blood cells, which have no nucleus (or DNA) and therefore cannot perform transcription. Our results show that transcription is not required for circadian oscillations in humans, and that non-transcriptional events seem to be sufficient to sustain cellular circadian rhythms. Using red blood cells, we found that peroxiredoxins, highly conserved antioxidant proteins, undergo ~24-hour redox cycles, which persist for many days under constant conditions (that is, in the absence of external cues). Moreover, these rhythms are entrainable (that is, tunable by environmental stimuli) and temperature-compensated, both key features of circadian rhythms. We anticipate that our findings will facilitate more sophisticated cellular clock models, highlighting the interdependency of transcriptional and non-transcriptional oscillations in potentially all eukaryotic cells.

At a glance

Figures

  1. Circadian oscillation of peroxiredoxin (PRX) oxidation in human RBCs.
    Figure 1: Circadian oscillation of peroxiredoxin (PRX) oxidation in human RBCs.

    a, RBCs from three human subjects (A, B, C) were kept under constant conditions (at 37°C, in total darkness) and sampled every 4h. b, RBCs incubated in alternating 12-h cycles of high (37°C) and low (32°C) temperature. Representative immunoblots showing oxidized/hyperoxidized peroxiredoxin (PRX-SO2/3) dimer are shown with loading controls. Quantification by densitometry is shown below. Values were normalized to the maximum for each blot. Solid line represents mean normalized intensity, with grey lines indicating s.e.m. boundaries. **P<0.01, ***P<0.001 by 1-way ANOVA (effect of time).

  2. Circadian rhythms of peroxiredoxin (PRX) oxidation are not affected by transcriptional and translational inhibition.
    Figure 2: Circadian rhythms of peroxiredoxin (PRX) oxidation are not affected by transcriptional and translational inhibition.

    RBCs were entrained under temperature cycles and then kept under constant conditions (at 37°C, in total darkness) and sampled every 4h. a, b, Representative immunoblots showing oxidized/hyperoxidized peroxiredoxin (PRX-SO2/3) dimer are shown for samples incubated with α-amanitin (a; α-AMN), or cycloheximide (b; CHX) for the entirety of the experiments. Quantification by densitometry is shown below. Values were normalized to the maximum for each blot. Each point represents a mean normalized intensity. NS, not significant; VEH, vehicle. Further details are shown in Supplementary Fig. 3.

  3. Temperature compensation of circadian peroxiredoxin oxidation rhythms.
    Figure 3: Temperature compensation of circadian peroxiredoxin oxidation rhythms.

    RBCs were entrained in temperature cycles (12h at 32°C, 12h at 37°C) for two complete cycles and then kept under a constant temperature of either 32°C or 37°C for the rest of the experiment and sampled every 4h as before. Immunoblots for oxidized/hyperoxidized peroxiredoxin (PRX-SO2/3) dimer from RBC lysates from subjects A, B and C are shown. Loading controls (Coomassie-stained gels showing haemoglobin monomer bands) for each blot are also shown. Quantification of the above immunoblots by densitometry is shown on the left of the figure.

  4. Expression patterns and oligomerization of peroxiredoxins.
    Figure 4: Expression patterns and oligomerization of peroxiredoxins.

    a, Immunoblots showing expression of the human peroxiredoxin paralogues (PRX1–PRX6) in RBCs and in mouse NIH3T3 cells. Loading of each lane was approximately equal. b, Oligomerization patterns of PRX and PRX-SO2/3 in RBCs. After two cycles of temperature entrainment, cells were kept under constant temperature (37°C) for the rest of the experiment, and sampled every 4h. Representative immunoblots for PRX2 and PRX-SO2/3 are shown. Whole blot images in Supplementary Fig. 5 illustrate the different oligomeric forms. c, d, Immunoblots were quantified by densitometry for PRX-SO2/3 (c) and PRX2 (d). Arrowheads indicate peaks of abundance.

  5. Circadian rhythms in haemoglobin oxidation and RBC metabolism.
    Figure 5: Circadian rhythms in haemoglobin oxidation and RBC metabolism.

    a, Intrinsic front-face fluorescence measurements of RBCs and controls. Experiments were performed under constant conditions (at 37°C, in total darkness). Mean values for each time point are shown (individual traces and further details are in Supplementary Fig. 6a). Two-way ANOVA (group × time) (***P<0.001). b, NADH and NADPH concentrations in RBCs. Mean values (±s.e.m.) for three experimental subjects are shown. One-way ANOVA (effect of time) for NADH/NADPH data was significant (***P<0.001). Two-way ANOVA (metabolite × time) did not reveal a significant difference between NADH and NADPH profiles. Individual profiles are shown in Supplementary Fig. 6b, c.

  6. Peroxiredoxin rhythms in nucleated cells.
    Figure 6: Peroxiredoxin rhythms in nucleated cells.

    a, Peroxiredoxin rhythms in mouse NIH3T3 fibroblasts synchronized by a serum shock. Immunoblots for Prx1, Prx6 and Prx-SO2/3 dimer are shown, in addition to Bmal1 and a β-actin loading control. b, c, Peroxiredoxin rhythms in mouse embryonic fibroblasts (MEFs). MEFs from wild-type or Cry1Cry2 double-knockout mice were entrained in temperature cycles and then kept under constant temperature (37°C) for the rest of the experiment (as shown in the schematic). b, Representative immunoblots of oxidized/hyperoxidized peroxiredoxin (Prx-SO2/3) dimer. c, Quantification of Prx-SO2/3 immunoblots by densitometry. Mean values (±s.e.m.) for n = 4 biological replicates are shown.

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Author information

Affiliations

  1. Department of Clinical Neurosciences, University of Cambridge Metabolic Research Laboratories, Institute of Metabolic Science, University of Cambridge, Addenbrooke’s Hospital, Cambridge CB2 0QQ, UK

    • John S. O’Neill &
    • Akhilesh B. Reddy

Contributions

A.B.R. and J.S.O’N. conceived, designed and performed the experiments, and wrote the manuscript.

Competing financial interests

The authors declare no competing financial interests.

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Supplementary information

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  1. Supplementary Information (4.2M)

    The file contains Supplementary Tables 1-2, Supplementary Figures 1-9 with legends and additional references.

Comments

  1. Report this comment #19385

    Paul Hartley said:

    In 1985, Radha et al., demonstrated a circadian rhythm of glutathione (GSH) levels over 48 hours in anucleate human platelets (1). It will be interesting to establish whether the platelet GSH rhythm represents the earliest example of a circadian clock in an anucleate, eukaryotic cell type.

    (1) Radha et al., 1985. Thrombosis Research 40; 823-831.

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