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Diurnal oscillations of endogenous H2O2 sustained by p66Shc regulate circadian clocks

Nature Cell Biology volume 21, pages 1553–1564 (2019)Cite this article

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Abstract

Redox balance, an essential feature of healthy physiological steady states, is regulated by circadian clocks, but whether or how endogenous redox signalling conversely regulates clockworks in mammals remains unknown. Here, we report circadian rhythms in the levels of endogenous H2O2 in mammalian cells and mouse livers. Using an unbiased method to screen for H2O2-sensitive transcription factors, we discovered that rhythmic redox control of CLOCK directly by endogenous H2O2 oscillations is required for proper intracellular clock function. Importantly, perturbations in the rhythm of H2O2 levels induced by the loss of p66Shc, which oscillates rhythmically in the liver and suprachiasmatic nucleus (SCN) of mice, disturb the rhythmic redox control of CLOCK function, reprogram hepatic transcriptome oscillations, lengthen the circadian period in mice and modulate light-induced clock resetting. Our findings suggest that redox signalling rhythms are intrinsically coupled to the circadian system through reversible oxidative modification of CLOCK and constitute essential mechanistic timekeeping components in mammals.

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Fig. 1: Endogenous H2O2 levels oscillate rhythmically in cells and in mouse livers.
Fig. 2: H2O2-sensitive TF screening showed that the redox state of CLOCK oscillates rhythmically.
Fig. 3: Cys 195 mediates the rhythmic oscillations in the redox state of CLOCK.
Fig. 4: Redox regulation of CLOCK at Cys 195 is essential for normal clock function.
Fig. 5: p66Shc is indispensable for robust oscillations of H2O2 levels and normal CLOCK function.
Fig. 6: p66Shc KO reprograms hepatic transcriptome oscillations and metabolic homeostasis.
Fig. 7: p66Shc modulates circadian behaviours in mice.

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Data availability

RNA-seq data that support the findings of this study have been deposited in the Sequence Read Archive (SRA) under accession code PRJNA449625. Previously published ChIP-seq data and crystal structure that were reanalysed here are available in the Gene Expression Omnibus (GEO) under accession code GSE39860 (ref. 36) and in the Protein Data Bank (PDB) under accession code 4F3L (ref. 26), respectively. Mass spectrometry data generated for the H2O2-sensitive TF screen and mass spectrometry data for purified CLOCK protein have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) through the iProX partner repository with the dataset identifiers PXD015265 and PXD015266, respectively. Source data are available online for Figs. 1b,c,e–g, 2e,g,i–k, 3b,c,g,l, 4c–e,g–n, 5a–k, 6a,b,f–k and 7b,c,e,g and Extended Data Figs. 1b,c, 2f–h, 3b,e, 4h,l, 5a,c,e,g–i,n–p, 6a,b and 7b,c. Unprocessed blots are provided in the Source Data. All other data supporting the findings of this study are available from the corresponding authors on reasonable request.

Code availability

Code used in this study (such as JTK_CYCLE for rhythmic analysis) are referenced in the Methods sections above. Any other code used in the study can be obtained from the corresponding authors on reasonable request.

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Acknowledgements

We thank X.-M. Xie and M. Lai (State Key Laboratory of Proteomics) for technical assistance and members of the De-Pei Liu laboratory for discussions about this project. This work was supported by grants from the Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences (CIFMS2017-I2M-1-008, 2019-RC-HL-006, 2016-I2M-1-015 and 2016-I2M-1-011), the National Natural Science Foundation of China (nos. 91849207, 81701387, 91639304, 31471126 and 31571193), the Medical Epigenetics Research Center, Chinese Academy of Medical Sciences (2017PT31035 and 2018PT31015) and Special Financial Grant from the China Postdoctoral Science Foundation (2017T100051). H.-Z.C. is also supported by the Youth Top-notch Talent Support Program and the Youth Yangtze River Scholar Program in China.

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Author notes

  1. Qian Gao

    Present address: Key Laboratory of Molecular Biophysics of Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China

  2. Na Liu

    Present address: College of Life Sciences, Hubei Key Laboratory of Edible Wild Plants Conservation and Utilization, National Demonstration Center for Experimental Biology Education, Hubei Normal University, Huangshi, China

  3. Ji-Min Cao

    Present address: Key Laboratory of Cellular Physiology, Ministry of Education, Department of Physiology, Shanxi Medical University, Taiyuan, China

  4. These authors contributed equally: Jian-Fei Pei, Xun-Kai Li, Wen-Qi Li.

Authors and Affiliations

  1. State Key Laboratory of Medical Molecular Biology, Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China

    Jian-Fei Pei, Xun-Kai Li, Wen-Qi Li, Yang Zhang, Xiao-Man Wang, Jia-Qi Fu, Shen-Shen Cui, Jia-Hua Qu, Xiang Zhao, De-Long Hao, Hou-Zao Chen & De-Pei Liu

  2. Department of Physiology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine, Peking Union Medical College, Beijing, China

    Qian Gao & Ji-Min Cao

  3. National Institute of Biological Sciences, Beijing, China

    Dapeng Ju, Na Liu & Eric Erquan Zhang

  4. Department of Chemistry, The Scripps Research Institute, Jupiter, FL, USA

    Kate S. Carroll

  5. State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences·Beijing, Beijing Institute of Lifeomics, Beijing, China

    Jing Yang

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Contributions

J.-F.P., H.-Z.C. and D.-P.L. conceived the project. J.-F.P., Q.G., Y.Z., X.-K.L. and W.-Q.L. performed mouse locomotor experiments and tissue collection. J.-F.P., X.-K.L., W.-Q.L., Y.Z., J.-Q.F. and S.-S.C. performed H2O2 assays, reporter assays, DCP-Bio1, BTD and BIAM labelling. Y.Z. and X.-K.L. constructed vectors and site-directed mutagenesis. X.-K.L. purified proteins and performed experiments about knockin mice. X.-K.L., W.-Q.L. and J.-F.P. performed EMSA and non-denaturing PAGE. J.-F.P. and X.-M.W analysed RNA-seq data. Y.Z., X.Z. and D.-L.H. performed Clock knockout mice experiments. J.Y. performed mass spectrometry and provided intellectual support for redox subject. J.Y. and K.S.C. provided technical support for redox modification examination. J.-F.P. performed real-time luciferase assays with help from D.J. and N.L. E.E.Z. conceived the LumiCycle design and provided intellectual support for the project. J.-F.P. prepared the illustrations and wrote the manuscript under the guidance of H.-Z.C. and D.-P.L. J.-H.Q. and J.-M.C. contributed to revision of characters. All of the authors contributed to data analysis and reviewed the manuscript. H.-Z.C. and D.-P.L. supervised the study.

Corresponding authors

Correspondence to Hou-Zao Chen or De-Pei Liu.

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Extended data

Extended Data Fig. 1 Endogenous H2O2 levels oscillate rhythmically in cells.

a, Time-lapse microscopy of circadian HyPerRed fluorescence in three individual cells for one day post-serum shock. N2a cells were transfected with HyPerRed, and images were obtained every 30 min (n = 3 independent experiments with similar results). b, Time-lapse microscopy of circadian HyPerRed fluorescence in three individual cells for one day without a serum treatment. U2OS cells were stably expressing HyPerRed, and images were obtained every 30 min. Note that the three cells have widely different phases (n = 3 independent experiments with similar results). c, Time-lapse microscopy of HyPerRed and GFP fluorescence under the same promoter in the same cell for one day without a serum treatment. U2OS cells were stably expressing HyPerRed and GFP, and images were obtained every 30 min (n = 3 independent experiments with similar results). Source data are provided in Statistics Source Data Extended Data Fig. 1.

Source data

Extended Data Fig. 2 H2O2-sensitive transcription factor (TF) screening identifies that the redox state of CLOCK oscillates rhythmically.

a, Reactive thiols of cysteine residues in proteins are oxidized to S-sulfenic acids (–SOH) by H2O2. Sulfenic acids are unstable and further react with proximal thiol groups to form disulfides, which are reversible oxidative modifications that can be restored to free thiols. b, Changes in the redox state of cysteine residues in proteins are monitored by detecting free thiols with biotin-conjugated iodoacetamide (BIAM) and by detecting cysteine sulfenic acid (R-SOH) with DCP-Bio1 or BTD. c, Representative western blot of S-sulfenylated CLOCK (CLOCK-SOH) from mouse livers labelled by DCP-Bio1. Biotin-blocked streptavidin-magnetic beads were used as a negative control. d, Representative western blot of free reactive thiols in CLOCK labelled by BIAM in vivo and in vitro from N2a cells. Biotin-blocked streptavidin-magnetic beads were used as a negative control. e, Representative western blot of free thiols of CLOCK labelled by BIAM from N2a cells treated with H2O2 (200 μM) for 0, 5, or 20 min and fractionated, followed by immunoprecipitation with CLOCK antibody. f, Relative levels of CCG transcripts in MEFs treated with H2O2 (200 μM) for 12 h. Data are presented as the means ± SEM (n = 3 independent biological samples). P values were calculated using an unpaired two-tailed Student’s t test. g, Relative HyPerRed intensity in U2OS cells stably expressing HyPerRed throughout the circadian cycle (n = 6 independent biological samples). h, Representative western blot of CLOCK-SOH labelled by DCP-Bio1 in U2OS cells over the circadian cycle. n = 3 independent experiments for c,d and n = 2 independent experiments for e,h with similar results. Source data are provided in Statistics Source Data Extended Data Fig. 2. Unprocessed blots are shown in Source Data Extended Data Fig. 2.

Source data

Extended Data Fig. 3 Cysteine195 mediates the rhythmic oscillations of CLOCK’s redox state.

a, Expression levels of each mutant CLOCK plasmid in HEK293T cells examined by immunoblotting (n = 2 independent experiments with similar results). b, Relative luciferase activities of Per1:Luc in HEK293T cells transfected with WT or C195S mutant CLOCK plasmids in the absence or presence of BMAL1 (n = 3 independent biological samples). Data are presented as the means ± SEM. c, Schematic of the target site at the Clock locus. In the double-stranded DNA, the sgRNA target is shown in blue and the PAM sequence is shown in red. Red arrowhead indicates the Cas9 cleavage site. In the donor DNA, the replaced nucleotides are shown in red (for point mutation) or blue (for silent mutation). d,e, Representative western blot (d) and quantification (e) of CLOCK-SOH labelled by DCP-Bio1 in MAFs from WT and ClockC195S mice for one circadian cycle (n = 2 independent experiments with similar results). Source data are provided in Statistics Source Data Extended Data Fig. 3. Unprocessed blots are shown in Source Data Extended Data Fig. 3.

Source data

Extended Data Fig. 4 Redox regulation of CLOCK at Cysteine195 is essential for normal clock function.

a, Schematic of the recombinant WT/C195S-CLOCK (amino acids 26–384) and BMAL1 (amino acids 62–447) proteins. b, Coomassie Brilliant Blue staining of the recombinant BMAL1 and WT/C195S-CLOCK proteins. c,d, Representative image of a non-denaturing polyacrylamide gel electrophoresis (PAGE) gel showing the heterodimer of recombinant BMAL1 and WT- or C195S-CLOCK treated with or without different concentrations of H2O2. e, Representative image of a non-denaturing PAGE gel of the heterodimer of the recombinant BMAL1 protein and increasing concentrations of the recombinant WT-CLOCK protein. f,g, Representative EMSA of recombinant BMAL1 or the heterodimer of recombinant BMAL1 and WT-CLOCK binding to increasing concentrations of the G-box probe (f) and the heterodimer binding to G-box probe treated with or without H2O2 (10−5 mM) (g). h, Relative mRNA levels of Bmal1 in WT and ClockC195S MAFs over the circadian cycles (n = 3 independent biological samples per time point). i, Schematic of 20-nt sgRNA target sequence of Clock (blue) and PAM (red). Red arrowhead indicates Cas9 cleavage site. j, Sequencing of PCR product from a Clock KO mouse. Black arrows indicate the location of mutations introduced by CRISPR/Cas9. k, Representative immunoblot of CLOCK in WT and Clock KO MAFs. l, ChIP analysis of WT- and C195S-CLOCK proteins binding to the Dbp promoter (-1360 to -1297) (-508 to -414) in N2a cells. The Rpl19 promoter served as the negative control (n = 3 independent biological samples). P values were calculated using one way ANOVA with a Bonferroni’s post hoc test. n = 2 independent experiments for b-g,j,k with similar results. Data are presented as the means ± SEM. Source data are provided in Statistics Source Data Extended Data Fig. 4. Unprocessed blots are shown in Source Data Extended Data Fig. 4.

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Extended Data Fig. 5 P66Shc is indispensable for robust oscillations of H2O2 levels and normal CLOCK function.

a, Relative H2O2 concentration in N2a cells transfected with a gradient mass of p66Shc (n = 3 independent biological samples). b, Representative immunoblot of p66Shc phosphorylation at Ser36 in MEFs treated with LY333531 (1 μM). c-i, Relative mRNA levels or representative immunoblot of p66Shc in MEFs (c,d), in livers (e,f), in N2a cells overexpressing CLOCK and BMAL1 (g), or in WT MAFs and Cry DKO MAFs (h,i) (n = 3 independent biological samples per group for c,e,g,h). j,k, Analysis of E-box elements on the promoter of mouse p66Shc (5 kb) (j) and its evolutionary conservation among multiple species (k). l, Representative immunoblot of reduced HA-CLOCK after transient transfection of p66Shc. m, Representative immunoblot of p66Shc in p66Shc KO MEFs rescued by p66Shc overexpression. n, Relative mRNA levels of CCGs in N2a cells overexpressing the WT or S36A mutant of p66Shc (n = 4 independent biological samples, except the Nampt in p66Shc group where n = 5, and the Per2 in p66Shc group and the Wee1 in p66S36A group where n = 3). o, Relative levels of Nampt mRNA in N2a cells overexpressing WT or S36A mutant of p66Shc and treated with H2O2 (200 μM) or catalase (1000 U/ml) (n = 5 independent biological samples). p, Relative amplitude of mPER2::LUC bioluminescence rhythms from WT and p66Shc KO liver explants (n = 9 independent biological samples). P values are shown for the comparisons to the control (a,g), to the first time point for p66Shc (c,e), to Cry DKO (h), to p66Shc (n), to p66S36A (o), and to WT group (p). P values were calculated using an unpaired two-tailed Student’s t test (n,p) and one-way ANOVA with a Bonferroni’s post hoc test (a,c,e,g,h,o). Data are presented as the means ± SEM. n = 3 independent experiments for b,d,f,i,m and n = 2 independent experiments for l with similar results. Source data are provided in Statistics Source Data Extended Data Fig. 5. Unprocessed blots are shown in Source Data Extended Data Fig. 5.

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Extended Data Fig. 6 P66Shc KO reprograms hepatic transcriptome oscillations.

a, Phase analysis of transcripts that oscillate only in the WT or p66Shc KO group. b, Phase analysis of genes that retain oscillations in both WT and p66Shc KO mice. RNA-seq analysis of the whole transcriptome was performed using total RNA obtained from three mouse livers each time point, which were pooled and then divided into two samples, at 4-h intervals for one circadian cycle under DD conditions. All analyses are from n = 1 RNA-sequencing experiment. Source data are provided in Statistics Source Data Extended Data Fig. 6.

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Extended Data Fig. 7 P66Shc modulates circadian behaviours in mice.

a, Representative western blot of p66Shc in mouse SCN and cortex at CT8 and CT14 under DD conditions. Three mouse SCNs or cortices were pooled per time point (n = 3 independent experiments with similar results). b, Relative levels of p66Shc mRNA in the SCN for one circadian cycle under DD conditions (n = 3 independent biological samples per time point). P values are shown for the comparisons to the first time point for p66Shc. Data are presented as the means ± SEM. c, Representative double-plot actograms of wheel-running activities and period lengths from two WT (left) and two p66Shc KO female mice (right) under DD conditions after LD entrainment. Red lines indicate the day on which DD conditions were initiated. P values are shown for the comparisons of p66Shc KO with WT. Values are presented as the means ± SEM (n = 9 mice). P values were calculated using one-way ANOVA with a Bonferroni’s post hoc test (b) and an unpaired two-tailed Student’s t test (c). Source data are provided in Statistics Source Data Extended Data Fig. 7. Unprocessed blots are shown in Source Data Extended Data Fig. 7.

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Supplementary information

Reporting Summary

Supplementary Table

Supplementary Table 1. TFs identified in control and H2O2-treated groups. Supplementary Table 2. Information of nucleotide sequences used in the study. Supplementary Table 3. RNA-seq results in WT and p66Shc KO mouse livers for one circadian cycle.

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Pei, JF., Li, XK., Li, WQ. et al. Diurnal oscillations of endogenous H2O2 sustained by p66Shc regulate circadian clocks. Nat Cell Biol 21, 1553–1564 (2019). https://doi.org/10.1038/s41556-019-0420-4

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