Regulation Of Circadian Rhythmic Oscillations Of Endogenous Hydrogen Peroxide On The Circadian Clock

Biological clock is a conservative timing mechanism in evolution,which enables organisms to predict the change of the environment,and adjust their behavior,physiological and biochemical reactions to adapt to the environment.The molecular mechanism of mammalian circadian rhythm is cell-autonomous transcription-translation feedback loops(TTFLs),and the main components of the core TTFLs include the positive regulatory elements,CLOCK and BMAL1,and the repressor complex formed by PER and CRY,which is the most important negative regulatory element,thus the positive and negative regulatory elements constitute a self-sustaining negative feedback loop together.However,the discovery of transcription independent oscillation,represented by the oxidation of peroxiredoxin(PRDX),has led to a rethinking of the molecular mechanism of circadian rhythm.However,it is still unclear whether redox system can directly participate in the regulation of circadian rhythm.In this study,we found that the redox modification of CLOCK mediated by endogenous H2O2 plays an important role on the biological function of CLOCK,thus establishing a new link between redox system and circadian rhythm.First,we found that the levels of endogenous H2O2 exhibited circadian oscillations in mammals,both in cultured cells and in mice livers.In order to find the direct coupling point between the oscillation of H2O2 and TTFLs,we used a screening strategy based on catTFRE-MS to identify the transcription factors that could sense the change of H2O2 in vivo.Interestingly,we found that the DNA-binding activity of CLOCK,the core operator of the circadian clock,could be significantly activated by H2O2.We also found that the redox state of CLOCK showed circadian oscillations in both cell lines and mouse livers,and the mRNA level of clock-controlled genes was also upregulated by the H2O2 treatment.Because the redox reaction is based on the oxidation and reduction of cysteine,so next we wanted to determine which cysteine residues were involved in the H2O2-mediated oxidative regulation of CLOCK.Then we did point mutation experiments and mass spectrometry experiments of the eight cysteine residues within the N-terminal region of CLOCK protein,the results showed that C195 was the most sensitive cysteine to H2O2.To determine whether Cl 95 mediates the rhythmic oscillations of the redox state of CLOCK in vivo,we constructed C195S knockin(ClockC195S)mice using CRISPR/Cas system and examined the redox state of WT-MAFs and Clock195s-MAFs respectively.Remarkably,the robust oscillations of CLOCK'S redox state,which were observed in WT-MAFs,were substantially impaired in ClockC195S-MAFs,suggesting that the redox modification of CLOCK by H2O2 is mediated by C195 of CLOCK.Next,we wanted to explore the effect of redox modification on the function of CLOCK.Based on the analysis of the crystal structure of CLOCK/BMAL1,we found that C195 is located in the PAS-A domain of CLOCK interacting with BMAL1 to form heterodimers in the three-dimensional structure.Therefore.we speculated that redox modification at C1 95 would affect the interaction between CLOCK and BMAL1.To verify our hypothesis,we did a series of biochemical experiments using WT-CLOCK,C195S-CLOCK,BMAL1 purified proteins in vitro.We found that H2O2 treatment could significantly enhanced the interaction between WT-CLOCK and BMAL1 and the binding ability between WT-CLOCK/BMAL1 and G-Box probe,but had no significant effect on C195S-CLOCK/BMAL1.In conclusion,the redox-sensitive Cys 195 on CLOCK can affect the dimerization formation of CLOCK/BMAL1 and its transcriptional activity.Moreover,compared with WT-MAFs,the transcriptional oscillation level and pattern of clock control genes(CCGs)in clock C195S-MAFS were changed.The detection of cell rhythm further confirmed the importance of redox sensitive 195 cysteine residue in biological rhythm.From the above findings,we provide a convincing demonstration supporting the notion that daily H2O2 oscillations are intrinsically linked to the circadian system through the reversible oxidative modification of CLOCK-Cysl95,which links the redox system to the molecular clock mechanism and constitutes the basic time keeping components together.These findings suggest important biological functions of the diurnal oscillations of endogenous H2O2 in the circadian rhythm of mammals,and a critical role for rhythmic CLOCK redox modification in bridging the gap between redox signaling oscillations and TTFLs.