Research ArticleCIRCADIAN RHYTHM

Chemical perturbations reveal that RUVBL2 regulates the circadian phase in mammals

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Science Translational Medicine  06 May 2020:
Vol. 12, Issue 542, eaba0769
DOI: 10.1126/scitranslmed.aba0769
  • Fig. 1 Discovery of cordycepin as a clock antiphasic compound.

    (A) Bioluminescent recordings of Per2-dLuc and Bmal1-dLuc U2OS cells upon cordycepin treatment (25 μM). Duplicate results were shown here, and DMSO treatment was used as control. (B) Effects of cordycepin (25 μM) and other indicated nucleosides on reporter rhythms in Per2-dLuc U2OS cells. For the other nucleosides, both 25 and 500 μM concentrations were tested. For clarity, the data shown here are representative of triplicate experiments (25 μM; n = 3); detailed analyses using the BioDare2 program are presented in fig. S2. (C) Chemical structures and effective doses of the only four antiphasic hits from a library consisting of 118 nucleosides. These four compounds are all adenosine analogs. (D) Effect of excessive adenosine (Adeno, 500 μM) on cordycepin (Cordy, 25 μM) on the circadian phase in Per2-dLuc U2OS cells. The dark lines of the traces represent the means, and the adjacent lighter areas indicate the SDs of the triplicate samples (n = 3). (E) Intracellular concentrations of cordycepin in treated U2OS cells (n = 3). Data are mean ± SD. (F) Left: Bioluminescent recordings of Per2-dLuc U2OS cells upon cordycepin treatment (25 μM) at indicated time. Data shown here are representative of triplicate experiments (n = 3). Right: A phase-responsive curve following cordycepin treatment. n = 4 for each time point; data are presented as mean ± SD. The full data and BioDare2 analysis results are in data file S1. Arrows (in A, B, D, and F) indicate the time when administration of drugs; CT0 was set at medium change; for the experiments carried out in a Lumicycle (B, D, and F), CT0 was 24 hours post-medium change.

  • Fig. 2 Regulation of the murine locomotor phase by peripheral administration of cordycepin.

    Top: Double plots show mouse locomotor assays for the phase advance (A) and phase delay (B) experiments. Bottom: Statistical analysis of the onset of locomotor activity of mice. Cordycepin (15 mg/kg) was intraperitoneally injected at ZT11, as indicated by arrows. Yellow regions represent the light phase, and gray areas the dark phase. Sample sizes and P values from one-way ANOVA are included in the figure. Data are mean ± SD.

  • Fig. 3 Identification of RUVBL2 as the cordycepin target that mediates the phase shift.

    (A) Effects of individual siRNA molecules against RUVBL2 or GFP on cordycepin-induced circadian phase shift in Per2-dLuc U2OS cells. For each curve, the data are mean ± SD (n = 3) and circadian time (CT) starts at the medium change. (B) Physical interaction between RUVBL2 and cordycepin was examined with biotinylated pull-down competition assays. Left, whole cell lysate; right, purified protein. (C) Immunofluorescence staining of the RUVBL2 protein in the mouse adult brain at CT12 (n = 5). Whole panel: Coronal section of the WT mouse brain staining with 4′,6-diamidino-2-phenylindole (DAPI) (blue) and an anti-RUVBL2 antibody (red); upper-left insert: zoom-in on the hypothalamus region. (D) Recombinant RUVBL2 pulled down with purified BMAL1 protein. Lanes shown in order from left to right: full-length of recombinant human RUVBL2 (FL); domain 2 deletion RUVBL2 variant (d2, lacking of the regulatory domain for the helicase activity); negative control enhanced green fluorescent protein (EGFP); glutathione S-transferase (GST)–tagged recombinant human BMAL1 protein; purified BMAL1 with RUVBL2 FL; purified BMAL1 with EGFP; and purified BMAL1 and the d2 RUVBL2 variant. (E) A Venn diagram (upper) and a heat map (lower) of ChIP-seq data showing the high rate of co-occupancy of chromatin loci (genome wide) by RUVBL2 and BMAL1 in U2OS cells. (F) Histograms from time-course ChIP-seq analyses of mouse liver. The analyzed liver clock-controlled genes (1444 genes in total) are from a previous report (31). (G) A UCSC (University of California, Santa Cruz) genome browser view of the liver Dbp locus showing the dynamic occupancies of BMAL1 and RUVBL2.

  • Fig. 4 Structural and biochemical analyses of RUVBL2 involvement in cordycepin phase-shift effects.

    (A) Cartoon presentation of RUVBL2 in complex with cordycepin 5′-triphosphate (CoTP). The green region representing the N-terminal segment of RUVBL2 (amino acids 23 to 41), which is otherwise flexible and hence invisible in the apo RUVBL2 crystal [Protein Data Bank (PDB) code: 6H7X], folds into the protein (light yellow) in the presence of CoTP (stick ball). In this figure, chain E of heterohexamer was used. The CoTP omitted electron density (weighted Fo-Fc, in purple blue) was countered at 2.0 σ. (B) Interactions between CoTP and the indicated amino acid residues of RUVBL2. Red C3 within the compound highlights the 3′-deoxylation site, which distinguishes cordycepin from the naïve adenosine. Dashed lines represent hydrogen bonds. (C) Microscale thermophoresis assay quantifying the RUVBL2-CoTP and RUVBL2-ATP interactions. Because the thermophoretic dynamics of fluorescent labeled RUVBL2 changes upon binding to a nonfluorescent ligand CoTP, resulting in different traces, the fluorescence of thermophoresis was normalized with two temperature stimulations (F norm). (D) Left: Effect of phosphorylated cordycepin on the interaction between the purified RUVBL2 (His-tagged) and BMAL1 (Flag-tagged) proteins. Right: Effect of D299Q mutant variant of RUVBL2 on RUVBL2 interaction with BMAL1. (E) Effect of adenosine kinase inhibitor (10 μM) on cordycepin (25 μM)–induced phase shift in Per2-dLuc U2OS cells (n = 4, CT0 is at 24 hours post-medium change). Data are presented as mean ± SD. (F) Effects of cordycepin on the circadian phases shift of Per2-dLuc U2OS cells ectopically expressing D299Q, A85T, or WT variant of RUVBL2 (n = 4). Data are presented as mean ± SD.

  • Fig. 5 Mass action kinetics modeling and experimental validation of cordycepin phase effects on E-box gene transcription.

    (A) Top: A mathematical simulation exploring relationships among phase-shift efficiency (heat map color bar), a compound’s induction or inhibitory effect on E-box gene transcription (y axis), and varying drug exposure durations (pharmacokinetics) of the compound (x axis), assuming either a net inductive (left) or net inhibitory (right) influence. Bottom: Proposed changes of the transcription strength for E-box genes corresponding to the net inductive or net inhibitory impact of a compound (transcription activity under the untreated condition is set as “1” or 100%). (B) Live imaging of PER2::LUC mice administered cordycepin by intraperitoneal injection at ZT2. Data are presented as mean ± SD (n = 5 per group; *P < 0.05 and **P < 0.001, one-way ANOVA). (C and D) Direct comparison of theoretical trajectories and experimental results of PER2 and BMAL1 gene expression upon cordycepin treatment administered at either the peak or the (C) or trough (D) of the Per2-dLuc signal intensity. For the mathematical simulation, the maximal gene expression under the untreated condition is set as 100%. Dashed blue lines indicate the free-run controls, and solid pink lines stand for the trajectories altered by the perturbations. Data are presented as mean ± SD from triplicate samples (n = 3).

  • Fig. 6 An RUVBL2-containing super-complex regulates the circadian phase.

    (A) A megadalton circadian super-complex was detected by tandem purification with anti-Flag and anti-HA antibodies, elution of the complex from beads, BN-PAGE, and silver staining. The band containing the complex (size ~1.4 MDa) was subsequently excised (dash-box) and digested with trypsin and analyzed via nano–liquid chromatography–MS/MS (nanoLC-MS/MS). Lanes from left to right: Flag-HA-Bmal1–rescued Bmal1−/− cells (B−/− + FH-B); Bmal1−/− only (B−/− only) cells as a negative control; and 10× dilution of the left lane amount. Color coding for the identified proteins: black, known core clock components; blue, previously reported clock-associated proteins; red, RUVBL2 or other putative clock-associated proteins in this circadian megadalton complex. (B) Effect of cordycepin treatment on the megadalton circadian repressive super-complex was analyzed with BN-PAGE and Western blot, with samples tandem purified from nuclear lysates of unsynchronized cells: Bmal1−/− only (B−/− only); Flag-HA-Bmal1–rescued (+FH-B); and Flag-HA-Bmal1–rescued cells treated with 25 μM cordycepin for 1 hour (+FH-B + Cordy). Another DNA binding helicase (CHD3) served here as a negative control. Arrows indicate the BMAL1-containing ~1.4-MDa super-complex. (C) Co-IP of HA-CRY1 with FLAG-BMAL1/FLAG-CLOCK transiently expressed in HEK293T cells, with either DMSO or cordycepin treatment. (D) Heat map views of spike-in ChIP-seq data showing the chromatin-bound intensities of BMAL1, RUVBL2, CRY1, and CRY2, with either DMSO or cordycepin treatment in unsynchronized U2OS cells. (E) The top 400 binding intensities of E-box loci for each protein were further quantified. (F) UCSC genome browser views of the binding of BMAL1, RUVBL2, CRY1, and CRY2 with (+) or without (−) cordycepin treatment. The representative E-box–containing genes shown here are PER1, PER2, DBP, and NR1D1.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/12/542/eaba0769/DC1

    Materials and Methods

    Fig. S1. Chemical screen for clock shifter compounds.

    Fig. S2. BioDare2 analyses of the various nucleotides including cordycepin for the clock effect.

    Fig. S3. Cordycepin hijacks the adenosine metabolism pathways to shift circadian phase.

    Fig. S4. Cordycepin regulates the clock phase in the tissue explants.

    Fig. S5. Cordycepin penetrates through the BBB but degrades quickly in the rodent brain.

    Fig. S6. The phase shift of cordycepin in mouse locomotor depends on the time of its administration.

    Fig. S7. Combined approaches reveal RUVBL2 as the target of cordycepin in regulating the clock phase.

    Fig. S8. Knockdown efficiency of RUVBL2 in U2OS cells.

    Fig. S9. Rhythmic expression of RUVBL2 in the SCN.

    Fig. S10. Two mouse Ruvbl genes are rhythmically expressed in vivo.

    Fig. S11. TIP60 complex is likely involved in the clock regulation.

    Fig. S12. The PAQosome complex may not be involved in the clock regulation.

    Fig. S13. Cell lines and the BMAL1 coimmunoprecipitation assays.

    Fig. S14. Structural analysis of RUVBL ATPases and phosphorylated cordycepin.

    Fig. S15. Pharmacokinetic analyses of cordycepin and CoTP in U2OS cells.

    Fig. S16. Physical interactions between RUVBL2 proteins and CoTP/ATP.

    Fig. S17. Cordycepin induces the expression of E-box genes in vivo.

    Fig. S18. The PAQosome complex may not be present in the circadian super-complex.

    Fig. S19. Cordycepin disrupts the interaction between RUVBL2 and BMAL1.

    Fig. S20. Cartoon of the proposed megadalton circadian repressive super-complex on an E-box containing clock gene.

    Data file S1. Phase responsive curve of the cordycepin treatment analyzed by BioDare2.

    Data file S2. Cotreatment of pentostatin and cordycepin analyzed by BioDare2.

    Data file S3. siRNA screen targets responsible for the cordycepin treatment effect.

    Data file S4. IP-MS to identify the biotinylated cordycepin-bound proteins.

    Data file S5. IP-MS to identify the megadalton super-complex components via BN-PAGE.

    Data file S6. Statistics of crystallization data collection and structure refinement.

    Data file S7. Primers used for RT-PCR/qPCR.

    Data file S8. All digital data used for generating figures.

    References (5162)

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. Chemical screen for clock shifter compounds.
    • Fig. S2. BioDare2 analyses of the various nucleotides including cordycepin for the clock effect.
    • Fig. S3. Cordycepin hijacks the adenosine metabolism pathways to shift circadian phase.
    • Fig. S4. Cordycepin regulates the clock phase in the tissue explants.
    • Fig. S5. Cordycepin penetrates through the BBB but degrades quickly in the rodent brain.
    • Fig. S6. The phase shift of cordycepin in mouse locomotor depends on the time of its administration.
    • Fig. S7. Combined approaches reveal RUVBL2 as the target of cordycepin in regulating the clock phase.
    • Fig. S8. Knockdown efficiency of RUVBL2 in U2OS cells.
    • Fig. S9. Rhythmic expression of RUVBL2 in the SCN.
    • Fig. S10. Two mouse Ruvbl genes are rhythmically expressed in vivo.
    • Fig. S11. TIP60 complex is likely involved in the clock regulation.
    • Fig. S12. The PAQosome complex may not be involved in the clock regulation.
    • Fig. S13. Cell lines and the BMAL1 coimmunoprecipitation assays.
    • Fig. S14. Structural analysis of RUVBL ATPases and phosphorylated cordycepin.
    • Fig. S15. Pharmacokinetic analyses of cordycepin and CoTP in U2OS cells.
    • Fig. S16. Physical interactions between RUVBL2 proteins and CoTP/ATP.
    • Fig. S17. Cordycepin induces the expression of E-box genes in vivo.
    • Fig. S18. The PAQosome complex may not be present in the circadian super-complex.
    • Fig. S19. Cordycepin disrupts the interaction between RUVBL2 and BMAL1.
    • Fig. S20. Cartoon of the proposed megadalton circadian repressive super-complex on an E-box containing clock gene.
    • References (5162)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft Excel format). Phase responsive curve of the cordycepin treatment analyzed by BioDare2.
    • Data file S2 (Microsoft Excel format). Cotreatment of pentostatin and cordycepin analyzed by BioDare2.
    • Data file S3 (Microsoft Excel format). siRNA screen targets responsible for the cordycepin treatment effect.
    • Data file S4 (Microsoft Excel format). IP-MS to identify the biotinylated cordycepin-bound proteins.
    • Data file S5 (Microsoft Excel format). IP-MS to identify the megadalton super-complex components via BN-PAGE.
    • Data file S6 (Microsoft Excel format). Statistics of crystallization data collection and structure refinement.
    • Data file S7 (Microsoft Excel format). Primers used for RT-PCR/qPCR.
    • Data file S8 (Microsoft Excel format). All digital data used for generating figures.

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