DNA Polymerase ?R137Q Promotes Methylation Clock Gene Per1 Promoter To Regulate Mouse Liver Biological Clock Homeostasis

Mammalian physiology and behavior are coordinated by intrinsic and exogenous circadian clock into rhythms that are synchronized with the 24 h solar day.Mammalian circadian clock system finely controls multiple physiological events,such as sleep/wake cycle,body temperature,blood pressure,endocrine,immune response as well as DNA damage and repair etc.Circadian rhythms therefore achieve temporal homeostasis of organisms via responding environmental stimuli at the molecular level by regulating gene expression so that particular physiological processes reach a peak every 24 h.DNA polymerase ? is a 39 kDa protein which consists of 335 amino acid residues with DNA polymerase(31kDa)and deoxyribose phosphatase(dRP lyase)activities(8kDa).Pol? is a key enzyme in DNA base excision repair(BER)and plays an important role in maintaining genome integrity and stability.BER is a highly-conserved pathway for DNA repairing after single-base loss,single-strand breaks and base damage,and lesions caused by a wide variety of exogenous and endogenous sources including environmental genotoxicants,oxidizing agents,and other common exposures such as ionizing and UV irradiation.Statistically.BER pathway responsible for repairing up to 20.000 endogenous lesions per cell per day.Studies revealed that BER prevented cells from propagating DNA damage caused by exposure to endogenous and exogenous sources.Aberrations and damages of this pathway have proven to be detrimental and are associated with human diseases,especially cancer.Studies on progeroid syndromes have suggested that the DNA damage response is involved in regulation of metabolic homeostasis and circadian clock.Two missense germline variants of Pol? have been identified in the human population.These are R137Q and P242R.Among the two DNA Pol? polymorphisms,R137Q is particularly interesting because the residue at position 137 locates in the helix 7 of POL? protein and forms salt bridges with other adjacent amino acid residues.More importantly,Pol? was reported to be methylated(in vitro and in vivo)by PRMT1 but the methylation was at amino acid residue 137.The amino acid substitution of Arg by Gln results in a net positive charge loss and might lead to significant alterations in biochemical and physiological properties of the enzyme.In addition,a recent research showed that R137Q significantly reduced polymerase activity and impaired interaction with PCNA.Pol? R137Q has lower polymerase activity resulting in defective BER,which might subsequently contribute to genome instability and cancer development.A growing number of studies have confirmed that DNA damage can reset circadian clock,thus we concluded that the impaired BER efficiency caused by Pol?R137Q mutation may lead to shift of circadian clock.In our study,we found that except for slight increase in food intake,there was no significant difference in metabolic phenotype between Pol? R137Q mice and wild type mice.In addition,data analysis from Clocklab revealed that Pol? R137Q mutant mice sufered from hyperactivity compared with wild type mice.Then,we sacrificed several Pol? R137Q mice and wild type mice at ZT14.Since liver is the largest metabolic organ,we we analyzed the transcriptional group of mice liver by RNA-sequencing.Interestingly,microarray analysis of liver revealed that numerous clock genes changed significantly,including Emall,Cryl,Id2,Per1,Dbp.Chrono and Rev-erb?.Besides,we detected clock genes expression in different tissues through q-PCR and Western blot.These data suggested that Pol? is an important regulator involved in the hepatic circadian clock.Results from circadian rhythmic q-PCR showed that Pol? affects the circadian clock in a liver-specific manner.However,the inner molecular mechanism through which Pol? affects hepatic clock remains unknown.The consensus emerging from recent studies is that removal of cytosine methylation in mammalian cells can occur by DNA repair.Besides,DNA methylation is commonly perceived as a stable modification and plays important roles in gene expression.Therefore,we hypothesized that the impaired BER activity caused by Pol? R137Q mutation could results in the failure of gene demethylation which leading to the decline of gene expression.Bisulfite sequencing PCR and Methylated DNA Immunoprecipitation Sequencing indicated that Pol? R137Q mutation decreases Per1 gene expression through impairing BER demethylation efficiency.To sum up,Pol? R137Q mutation affects hepatic circadian rhythm through increasing methylation level of the fourth CpG island on the promoter of Per1.