ATM Regulates Biological Rhythms Through The Period2 Protein

Objective: This project aims to explore the regulation of circadian rhythms by DNA repair machinery.Methods:1.The oscillation of ATM protein was examined by Western blotting in immortalized MEF and HEK293 cells after synchronization.2.ATM KO HEK293 cells were generated by the CRISPR-Cas9 system.RT-PCR analysis of rhythmicity of circadian clock genes in MEF and HEK293 cells lacking ATM gene.3.Effects of ATM deficiency on circadian rhythms in mice were determined by running wheel test.4.The protein level of core clock genes in different cell lines and mouse tissues deficient for ATM were measured by Western blotting.5.Endogenous and exogenous PER2 protein degradation rate in ATM KO cells was assayed by CHX-chase analysis.6.Interaction between ATM and PER2 were determined by co-immunoprecipitation.7.Western blotting analysis of phosphorylation of Myc-PER2 in cells lacking ATM or treated with ATM inhibitors or activators.8.Mass spec analysis of potential phosphorylation sites of PER2 mediated by ATM.Result: 1.ATM protein level oscillates in both synchronized MEF and HEK293 cells.2.Disrupted circadian gene expression in both Atm KO MEFs and ATM KO HEK293 cells.3.Disrupted circadian rhythm in Atm+/-mice.4.ATM deficiency decreases PER2 level in both cells and mouse tissues.5.Loss of ATM accelerates PER2 degradation.6.Co-immunoprecipitation revealed potential interaction between ATM and PER2.7.Western botting showed that PER2 phosphorylation level decreased in ATM-KO cells,inhibition of ATM kinase activity by ku55933 accelerated PER2 degradation,and activation of ATM by CPT enhanced protein stability of PER2.8.Using proteomics strategy and high-resolution LC-MS/MS,we found a series of phosphorylation sites of Myc-PER2 from WT and ATM KO HEK293 cells.18 phosphorylation sites were identified in Myc-PER2 purified from WT cells and 16 phosphorylation sites were identified in Myc-PER2 purified from ATM KO cells.Conclusion: ATM protein level oscillates in immortalized MEF cells and HEK293 cells after synchronization.ATM regulates circadian rhythms probably by phosphorylating and thus stabilizing PER2.