A short-term heat shock-pulse resets circadian <i>mPer2</i> rhythms in NIH-3T3 fibroblasts.

<p>(<b>A</b>) Determining the optimal duration for the heat shock (HS) treatment to induce circadian <i>mPer2</i> expression. NIH-3T3 fibroblasts stably expressing <i>mPer2</i> promoter-driven luciferase (<i>mPer2</i>-Luc) and 3×HSE-driven SLR luciferase (HSE-SLR) were treated with different durations of HS as indicated. Circadian <i>mPer2</i> (blue) and HS element (HSE) (red) profiles were monitored by a real-time dual-color bioluminescence assay. Relative acute (RLU) and normalized (deviation from moving average) circadian profiles from each experiment (average values from 5 independent experiments) are shown. (<b>B</b>) NIH-3T3 cells stably expressing <i>mPer2</i>-Luc were treated with or without an HS pulse (43°C, 30 min) or dexamethasone (Dex). The acute <i>mPer2</i>-Luc elevation pattern in the HS-stimulated cells is quite similar to the pattern shown in panel <b>A</b>.</p

Heat shock factor 1 (HSF1) interacts with BMAL1:CLOCK after the heat shock (HS) pulse.

<p>Wild-type (WT) mouse embryonic fibroblasts (MEFs) were treated with or without an HS pulse. At 24 h after the HS pulse, the lysates were subjected to immunoblotting for PER2, HSP70, BMAL1, HSF1, and actin. Representative images from triplicate independent experiments are shown (<b>A</b>). WT and <i>Hsf1</i>^−/− MEFs were treated with the HS pulse. At the indicated times after the HS pulse, the lysates (<b>B</b>) and BMAL1/HSF1 immunoprecipitates (<b>D; CoIP</b>) were subjected to immunoblotting for PER2, HSP70, BMAL1, HSF1, CLOCK, and actin. Representative images from triplicate independent experiments are shown. Normalized PER2 and HSP70 protein levels (<b>B</b>; at 26 h after the HS pulse), and BMAL1 coimmunoprecipitated with HSF1 and CLOCK (<b>D</b>) are shown as average values from triplicate independent experiments. Error bars indicate standard deviation (SD) (*** P<0.001). (<b>C</b>) WT and <i>Hsf1</i>^−/− MEFs were treated with the HS pulse. At the indicated times after the HS pulse, the lysates were subjected to immunoblotting for PER2, HSP70, BMAL1, HSF1, and actin. Representative images from triplicate independent experiments are shown. Normalized circadian PER2 and HSP70 protein profiles plotted with average values from triplicate independent experiments are shown and error bars indicate SD.</p

Determining the heat shock elements (HSEs) required for the heat shock (HS)-synchronized circadian <i>mPer2</i> rhythms.

<p>(<b>A</b>) Prediction of HSEs adjacent to the E-box elements (shown as quadrangles) in the <i>mPer2</i> promoter and a design for their mutagenesis (HSE Mt1 and Mt2) are shown. (<b>B, C</b>) NIH-3T3 cells transfected with <i>mPer2</i>-Luc and wild-type (WT) HSEs, HSE Mt1 and Mt2 were treated with the HS pulse or (<b>C</b>) dexamethasone (Dex). Circadian <i>mPer2</i> profiles were monitored by the real-time bioluminescence assay. Relative acute (RLU) and normalized (deviation from moving average) circadian profiles from each experiment (average values from 5 independent experiments) are shown.</p

A CRY-based dual negative feedback model integrating the BMAL1– P-BMAL1 loop with the circadian core oscillator.

<p>Proposed model for CRY-CK2β–mediated circadian rhythms in BMAL1 phosphorylation by CK2α (the BMAL1–P-BMAL1 loop) as an integral part of the circadian core oscillator. In short, upon phosphorylation of BMAL1-S90 by CK2α (<b>step I</b>), BMAL1 binds to CLOCK (<b>step II</b>) to form a transcriptional activator complex for transcription of E-box promoter containing clock genes (i.e., <i>Cry</i> and <i>Per</i> genes) and clock-controlled genes (<b>step III</b>). CK2α remains bound to BMAL1 in a catalytically active state (as indicated by the red color). After a delay, CRY proteins will bind to the CLOCK–BMAL1–CK2α complex to inhibit E-box gene transcription (<b>step IV</b>). Upon dissociation of the BMAL1–CLOCK–CRY–CK2α complex from the DNA, CRY is released from the complex to allow CRY–CK2β binding, and subsequent BMAL1–CK2β binding, resulting in the formation of BMAL1–CLOCK–CRY–CK2β–CK2α complex (<b>step V</b>). This step, triggered by acetylation of BMAL-K537, renders CK2α inactive (as indicated by the grey color). After dissociation of the BMAL1–CLOCK–CRY–CK2β–CK2α complex, BMAL1 is degraded and/or dephosphorylated and deacetylated (<b>step VI</b>) to start a new cycle. For a detailed description of the model, see the <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002293#sec010" target="_blank">Discussion</a> section. For simplicity, PER proteins have not been included in the model and CRY1 and CRY2 proteins are collectively shown as CRY.</p

BMAL1-S90 phosphorylation is prerequisite for BMAL1-K537 acetylation and subsequent recruitment of CRY to BMAL1.

<p>(A) <i>Bmal1</i>^−/− MEFs stably expressing <i>mBmal1</i> promoter-driven Myc-BMAL1-WT (wild type), Myc-BMAL1-S90A (CK2−phosphorylation site-deficient mutant), and CMV-promoter-driven Myc-GFP (control) were Dex-synchronized. The supernatants from the cells harvested 22 h after treatment were subjected to Myc-BMAL1 IP. The Myc-BMAL1 IPs and total lysates (Load) were subjected to IB analysis for Acetyl-BMAL1 and BMAL1- co-immunoprecipitated (Co-IP) CRY1/2 and CLOCK. (B) Normalized Acetyl-BMAL1 and BMAL1-bound CRY1/2 and CLOCK levels are shown in the graph (<i>n</i> = 3 experiments) and error bars indicate SD.</p

CK2-mediated circadian BMAL1-Ser90 phosphorylation regulates the central and peripheral clocks.

<p>(A) Clock performance and BMAL1 phosphorylation in NIH-3T3:Per2L cells after treatment with BMs90p (a 14 amino acid BMAL1 peptide containing S90; concentrations as indicated) for 30 min and subsequent clock-synchronization by dexamethasone (Dex) treatment for 30 min. (a) Cell cultures were monitored for luciferase activity by real-time bioluminescence assay. Representative raw (left) and detrended/averaged (right) profiles are shown (<i>n</i> = 4). (b) Immunoblot (IB) analysis of BMAL1-immunoprecipitates (IP) and lysates from BMs90p-treated cells for BMAL1, P-BMAL1-S90 (Ser90-phoshorylated BMAL1), CK2α, CK2β, and actin (control). Shown are representative images of triplicate experiments. (c) Quantification of P-BMAL1-S90 levels in BMs90p treated cells (<i>n</i> = 3). Values in untreated cells were set as 1. Error bars indicate standard deviation (SD). (B) Clock performance of organotypic slices of liver and SCN from <i>mPER2</i>^<i>Luc</i> mice following treatment with 6 μM BMs90p or mock treatment with fresh medium around the PER2::LUC trough phase (liver; ~CT [Circadian Time] 5, SCN; ~CT2). (a) Luciferase activity was monitored by real-time bioluminescence imaging. Note the recovery of BMs90P dampened rhythms by medium change. (b) Quantification of rhythm amplitude and peak bioluminescence after BMs90p treatment (<i>n</i> = 5), in which values in untreated slices are set as 1. Error bars indicate SD. (C) Clock performance of small cell clusters in organotypic SCN slices from PER2::LUC mice following treatment with 6 μM BMs90p around the trough phase (~CT2). (a) Luciferase activity was monitored by real-time bioluminescence imaging (<i>n</i> = 24). Shown are detrended (colored lines) and averaged values (dotted line). (b) Representative examples of bright field (BF) and Per2L images at CT14 (around the peak phase) pre- and post-BMs90p treatment (day 2 and 4 respectively).</p

CRY-enhanced circadian oscillation of BMAL1–CK2β association in living cells.

<p>(A) Graphic representation of ELucN-CK2β, McLuc1-BMAL1, and ELucC-BMAL1 vectors for real-time bioluminescence imaging of CK2β-BMAL1 interactions as monitored by the Split Luc complementation system. IB analysis with anti-BMAL1 and CK2β antibodies reveals ectopic expression of His-tagged ELucC-BMAL1 (~100 kDa) and ELucN-CK2β ELucN-CK2β (~70 kDa) in Cos7 cells. (B) Cos7 cells were transfected with ELucN-CK2β and ELucC-BMAL1 (2.5 μg), and either Myc-HA-CRY1, Myc-CRY2, or (control) pcDNA (−) (5 μg). The presence of ectopically expressed CRY1/2 and native CK2α was confirmed by IB analysis (lower panel). BMAL1–CK2β binding was monitored by real-time bioluminescence imaging and plotted as the average value of peak bioluminescence levels (<i>n</i> = 5 experiments; upper panel). Error bars indicate SD. Note that BMAL1–CK2β binding was significantly facilitated by CRY1 and CRY/2. (C) Circadian rhythms in BMAL1–CK2β binding in Dex-synchronized U-2OS cells were monitored by real-time bioluminescence imaging of Split Luc activity (upper panel). The circadian profiles (Period = 25.3 h, Acrophase = 15.1 h) (<i>n</i> = 5; blue line) are shown with normalized values to the maximum value that was set as 1. Expression of McLuc1-BMAL1, native BMAL1, ELucN-CK2β, and native CK2β was confirmed by IB analysis (upper panel, insets). The circadian profile of P-BMAL1-S90 levels (representative examples of <i>n</i> = 3 experiments) was determined by IB analysis (lower panel) with anti-P-BMAL1-S90), quantified and normalized to actin content. Maximum values were set as 1 (upper panel: dotted red line). Error bars indicate SD.</p

BMAL1-K537 acetylation reduces BMAL1-S90 phosphorylation via enhancing BMAL1–CK2β association.

<p>(A) Reduced CRY-enhanced CK2β binding to mutant BMAL1-K537R, lacking a CLOCK-mediated acetylation site. As in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002293#pbio.1002293.g005" target="_blank">Fig 5</a>, except that an acetylation mutant ELucC-BMAL1 vector (K537R) was used. (B) Reduction of CK2-mediated BMAL1-S90 phosphorylation in SIRT1KO (<i>Sirt1</i>^<i>-/-</i>) cells, lacking the major clock-regulating deacetylase SIRT1. (a) Co-immunoprecipitation experiments with WT and SIRT1KO MEFs 23h after Dex treatment. IB analysis of BMAL1-IP and lysates for P-BMAL1-S90, CK2β, acetyl-BMAL1, SIRT1, BMAL1, and actin. Shown are representative examples of <i>n</i> = 3 experiments. (b) Quantification of P-BMAL1-S90 and BMAL1-bound CK2 β levels. Values in WT MEFs are set as 1. Error bars indicate SD.</p

CRY regulates CK2-mediated BMAL1-Ser90 phosphorylation.

<p>(A) Co-immunoprecipitation experiments with non-clock synchronized wild type (WT) and CRYdKO (<i>Cry1</i>^<i>-/-</i><i>/Cry2</i>^-/-) mouse embryonic fibroblasts (MEFs), and CRYdKO MEFs stably expressing <i>mCry1</i> promoter-driven Myc-CRY1 or CMV-promoter-driven Myc-GFP (control). IB analysis of BMAL1-, CK2α-, and CK2β-IP and lysates for BMAL1, P-BMAL1-S90, CK2α, CK2β, CRY1, CRY2, and actin (control). Shown are representative examples of <i>n</i> = 3 experiments. Red arrows indicate the position of major P-BMAL1-S90 (~90 kDa). (B) Quantification of P-BMAL1-S90 and CK2β–BMAL1 levels. Values in WT MEFs are set as 1. Error bars indicate SD. (C) In vitro analysis of CK2α-mediated BMAL1-S90 phosphorylation. Mixtures of CK2α and CK2β subunits and GST-BMAL1 were subjected to an in vitro kinase assay. BMAL1-Ser90 kinase activity was measured by IB analysis with anti-P-BMAL1-S90 (representative examples of <i>n</i> = 3 experiments), and plotted against CK2β/ (GST-) BMAL1 ratio. Values were normalized against activity in the absence of CK2β, which was set as 1. Error bars indicate SD. Note the inhibition of CK2α-mediated BMAL1-S90 phosphorylation by CK2β.</p

ROS Stress Resets Circadian Clocks to Coordinate Pro-Survival Signals

<div><p>Dysfunction of circadian clocks exacerbates various diseases, in part likely due to impaired stress resistance. It is unclear how circadian clock system responds toward critical stresses, to evoke life-protective adaptation. We identified a reactive oxygen species (ROS), H₂O₂ -responsive circadian pathway in mammals. Near-lethal doses of ROS-induced critical oxidative stress (cOS) at the branch point of life and death resets circadian clocks, synergistically evoking protective responses for cell survival. The cOS-triggered clock resetting and pro-survival responses are mediated by transcription factor, central clock-regulatory BMAL1 and heat shock stress-responsive (HSR) HSF1. Casein kinase II (CK2) –mediated phosphorylation regulates dimerization and function of BMAL1 and HSF1 to control the cOS-evoked responses. The core cOS-responsive transcriptome includes CK2-regulated crosstalk between the circadian, HSR, NF-kappa-B-mediated anti-apoptotic, and Nrf2-mediated anti-oxidant pathways. This novel circadian-adaptive signaling system likely plays fundamental protective roles in various ROS-inducible disorders, diseases, and death.</p> </div