Functional Investigation Of H4s47 O-GlcNAcylation In DNA Replication

DNA replication is fundamentally important for the duplication and inheritance of genetic information during cell division.To guarantee the precise duplication of the entire genome,DNA replication that is a highly regulated process is launched from multiple sites of the eukaryotic genome.These discrete sites are defined as replication origins whose licensing and activation underlie the initiation process of DNA replication.Intriguingly,large excessive origins are licensed but only a small fraction of origins is activated in each cell cycle.It still remains elusive how eukaryotic replication origins are selective activated.Although origins in yeast possess certain DNA feature,consensus DNA sequence that could comprehensively and precisely predict replication origins in mammals has not been identified.It has been shown that histone H4 modifications,including methylation and acetylation,play important roles in the control of replication origin licensing.Recent investigations revealed that histone H4 could also bear O-GlcNAcylation,a unique type of modification known as nutrition"senser",which is capable of sensing changes in the nutrition environment.Hitherto,only one O-GlcNAcylation site on H4,namely Serine 47（H4S47）,has been identified,but its biological function is still unknown.In this study,we combined site-directed mutation strategy with a series of functional assays,such as mass spectrometry,DNA fiber assay,chromatin-bound extraction and chromatin immunoprecipitation,to characterize the role of H4S47 O-GlcNAcylation in DNA replication.First,we validated H4S47 O-GlcNAcylation with mass spectrometry.After mutating H4S47 to alanine（Ala,A）that could not be O-GlcNAcylated,we evaluated the function of H4S47 O-GlcNAcylation in DNA replication.By immunofluorescence,flow cytometry and DNA fiber assay,we found that the mutation of H4S47 significantly inhibited DNA replication and hindered replication initiation.It appears that O-GlcNAc transferase OGT enriches on chromatin in S phase and resides at replication sites.Importantly,it enhanced DNA replication by catalyzing H4S47 O-GlcNAcylation.Further,unbiased interactome analysis revealed that wild-type H4(H4^WT)is favored over mutant H4^S47A for association with helicase mini-chromosome maintenance（MCM）complex,supporting an important role for H4S47 O-GlcNAcylation in replication initiation.Because replication initiation consists of origin licensing and activation,which can be reflected by chromatin-loading and phosphorylation of MCM complexes respectively,we next evaluated the impact from H4S47 mutation on these two steps.Instead of MCM loading on chromatin,the phosphorylation of MCM2（MCM2 p S53）was impaired upon H4S47 mutation,arguing that H4S47 O-GlcNAcylation may play a critical role in origin activation.Given that both CDK（cyclin-dependent protein kinase）and DDK（DBF4-dependent protein kinase）are crucial for the control of origin activation,we continued to explore whether they were involved in the regulation of H4S47 O-GlcNAcylation-dependent origin activation.H4S47 mutation markedly impaired DDK association on chromatin,but failed to affect CDK binding.In parallel,in vitro GST-pulldown assay revealed that H4S47 O-GlcNAcylation could promote the formation of H4-DDK-MCM2 ternary complex.Also,we verified the important function of H4S47 O-GlcNAcylation in DNA unwinding by detecting the accumulation of single-stranded DNA（ss DNA）associated with origin activation,Finally,our re-Ch IP assay confirmed the"H4S47 O-GlcNAcylation-MCM2 p S53"regulatory axis at the established origins in human cells.Together,we demonstrate that H4S47 O-GlcNAcylation regulates origin activation by directing DDK recruitment on chromatin,which in turn facilitates the activation of MCM complex and thereby replication initiation.Our results unveil a novel function for histone O-GlcNAcylation and illustrate a previously unappreciated mechanism for the regulation of eukaryotic DNA replication.Together,these findings would be useful for us to understand the spatio-temporal control of DNA replication in higher eukaryotes.