Mechanism Of Histone Methylation Modification H4R3me2s In DNA Oxidative Damage Repair

In the course of life,cells are subjected to oxidative stresses caused by endogenous and exogenous factors,such as chemical agents,radiation and cell normal metabolisms,which can lead to produce excessive ROS.Previous studies have shown that ROS can directly attack DNA,which will change the structure of the DNA molecule and induce oxidative DNA damage,a the formation of 8-hydroxydeoxyguanine by oxidation of the position eight of carbon atom of guanine is the most common,if not repaired,it will lead to instability of the genome and even cause cancer.In the cellular DNA repair networks,Base Excision Repair(BER)is the primary pathway responsible for DNA oxidative damage repair,8-oxoguanine DNA glycosylase 1(OGG1)plays an important role in this repair pathway,it can specifically recognize and remove 8-hydroxydeoxyguanine,and is essential to maintain the stability of the genome.In addition,Flap endonuclease-1(FEN1)is also a key protein in this repair pathway,it is a structure-specific nuclease,which can specifically remove the structure of 5 '-flap from the DNA strand.It is vital for DNA repair,the maturation of Okazaki fragment and the maintenance of telomere.In eukaryotes,DNA is packaged as chromatin,nucleosomes are the basic structure of chromatin,which is packaged by a core histone and a 146bp DNA.The histone N-terminus is located outside of the nucleosome structure and is rich in positively-charged amino acids that can undergo a variety of covalent modifications,methylation is an important post-translational modification,which mainly occurs at lysine and arginine residues.In recent years,arginine methylation has attracted more and more attention,which is catalyzed by PRMTs in mammalian cells.H4R3me2s,that is symmetrical dimethylation modification at the position third of arginine on histone H4,has been reported that it is closely associated with the transcriptional regulation,but little is known about the role on DNA damage repair.To explore whether H4R3me2s play roles in DNA damage repair,and what the specific mechanism is,we performed the following experiments.First of all,we treated HeLa cells with DNA oxidative damage reagent H2O2,the western blotting results showed that H4R3me2s had a significant increase,and the same results were observed in immunofluorescence,which indicated that H4R3me2s may be involved in DNA oxidative damage repair.Next,we performed pull-down assay with biotin-labeled nucleic acid probes and found that H4R3me2s had stronger affinity with probes with 8-oxodG.As we know,OGG1 can specifically recognize 8-oxodG,so we guessed that there is a certain link between OGG1 and H4R3me2s.We overexpressed and knocked down OGG1 in cells,respectively,and the results indicated that level of H4R3me2s was positively correlated with OGG1,and the up-regulation of H4R3me2s after H2O2 treatment was significantly reduced in OGG1-deficient cells,the results implied that OGG1 participated in the formation of H4R3me2s.H4R3me2s modification is the product of PRMT5,so we tested whether OGG1 can affect the function of PRMT5,the results indicated that depletion of OGG1,while not affecting the expression level of PRMT5,dramatically reduced the interaction between PRMT5 and H4R3mel,which implied that OGG1 plays important role in PRMT5-mediated the formation of H4R3me2s.We also confirmed that OGG1 can interact with PRMT5 in vitro and in vivo by Co-IP assay.Taken together,we suggested that OGG1 can regulate the level of H4R3me2s through interacting with PRMT5.Next,to explore the biological role of the increase of H4R3me2s after H2O2 treatment,we performed a pull-down assay with the biotin-labeled H4R3me2s peptide,and then performed a mass spectrometry analysis of the pull-down mixture.The results showed that FEN1 can interact with H4R3me2s.To further confirm the results,we performed in vitro and in vivo Co-IP and immune co-localization experiments.The results indicated that there is indeed an interaction between FEN1 and H4R3me2s,and the combination will be enhanced after H2O2 treatment.FEN1 is a nuclease,to investigate the effect of H4R3me2s on the enzymatic activity of FEN1,we performed the FEN1 nuclease assays in vitro.The results showed that H4R3me2s significantly enhanced FEN1 activity,and the subsequent LP-BER assays further confirmed this result.Meanwhile,we also demonstrated that H4R3me2s can enhance the affinity of FEN 1 to its substrate through EMSA assay,which indicated that H4R3me2s can enhance the activity of FEN1 by enhancing its affinity to its substrates.To further illustrate the role of H4R3me2s on DNA repair,we knocked down PRMT5 and overexpressed histone H4,respectively,to change the level of H4R3me2s in vivo and then tested DNA damage in cells,and the results showed that reducing the level of H4R3me2s can aggravate the DNA oxidative damage and even increase the sensitivity of cells to H2O2,but when we increased the level of H4R3me2s,the DNA damage was retarded,and also enhanced the resistance to H2O2.In summary,our data supported that histone arginine methylation modification H4R3me2s had a significant up-regulation under the regulation of OGG1 and PRMT5 when cells were subjected to oxidative stress,and the increased level of modification will enhance the enzymatic activity of the downstream DNA repair enzyme FEN1 and promote oxidative DNA damage repair.Therefore,histone arginine methylation modification H4R3me2s can be recognized as a "reader" protein to promote subsequent DNA damage repair.