Functional Investigation Of O-GlcNAc Modification Of Histone H3 And H4 In DNA Replication

The accurate and efficient duplication of the entire genome is fundamentally important for the development of diverse organisms.Since DNA is replicated in the context of chromatin,the organization and re-organization of chromatin structure are thus constantly needed throughout the replication process.Besides chromatin remodelers and modifiers,histone modifications including acetylation and methylation have been shown to be important for DNA replication.Interestingly,recent work has demonstrated that O-GlcNAc modification can also take place on histones.However,O-GlcNAcylation sites on histones and their responsible function in DNA replication remain unclear.In this study,we first used inhibitors to up-regulate or down-regulate the level of O-GlcNAc modification in human osteosarcoma U2 OS cells,which were pulse-labeled by Ed U prior to the analysis of DNA replication efficiency by flow cytometry.It appeared that the down-regulation of O-GlcNAcylation reduced replication efficiency(Ed U positive cells).Under the same condition,S phase progression was delayed revealed by our synchronization assay.These results suggest that O-GlcNAcylation of proteins in cells is required for efficient DNA replication as well as S phase progression.In agreement with previous studies,we also observed clear O-GlcNAc modification in histone H3 and H4 by using a click-i T?O-GlcNAc enzymatic labeling system.Furthermore,we performed site-directed mutagenesis to mutate potential O-GlcNAc modification sites on histone H3 and H4,which were implicated by mass spectrometry data,for the characterization of O-GlcNAc modification site(s).Interestingly,the level of O-GlcNAcylation on H3 and H4 was reduced,when H3T32 and H4S47 were mutated.Importantly,DNA replication was impaired in cells expressing H3T32 and H4S47 as compare to control cells.We thus conclude that H3T32 and H4S47 are the O-GlcNAc modifications sites and probably play an important role in DNA replication.Together,our work will contribute to the functional dissection of histone O-GlcNAcylation in DNA replication,and provide a useful tool for the mechanistic investigation of post-translational modifications on histones in the regulation of genome stability.