OGT1-mediated O-GlcNAcylation Of Thr755 Of NSL3 Regulates The Holoenzyme Activity Of MOF/NSL Complex

Based on the traditional concept of genetics,the meaning of epigenetics has been continuously updated in constant study of the molecular mechanism of eukaryotic gene expression regulation.The current definition is about studying the genetic function and phenotypic changes in organisms without changing the genome nucleotide sequence during mitosis and meiosis.Epigenetic regulation mainly includes chromatin remodeling,DNA methylation,histone modification,and non-coding RNA.This article mainly discusses the molecular mechanism of the holoenzyme activity of histone acetyltransferase MOF/ NSL complex regulated by OGT mediated O-GlcNAc modification occurs on NSL3 Thr755.MOF(males absent on the first)/ NSL histone acetyltransferase complex has a wide range of substrate specificities.In addition to acetylating H4K16,it can also acetylate H4K5 and K8 in recombinant chromatin in vitro.What's more,non-histone p53 is one of its substrates.As the only O-GlcNAc transferases,OGT catalyzes the adding of O-GlcNAc groups to Ser/Thr residues of substrate proteins,whereas OGA catalyzes the reverse reaction to remove the O-GlcNAc groups from proteins.This makes O-GlcNAc modification a inducible,reversible,dynamic post-translational modification.OGT is involved in regulating large variety of cellular processes,such as gene transcription,cell cycle progression,apoptosis,and cell signaling.The broken balance of intracellular O-GlcNAcylation levels can cause a variety of diseases including diabetes,cancer,neurodegenerative diseases and cardiovascular diseases.OGT is frequently assembled into different protein complexes as a component,regulating the functions and other epigenetic modifications of substrate proteins,thus coordinate specific biological functions.NSL3 is another essential subunit of MOF/NSL histone acetyltransferase complex,which was reported to bind directly to OGT1 and be O-GlcNAc modified by OGT1.Except for increasing the protein stability,O-GlcNacylation of NSL3 could influence the global level of H4K16 Ac,H4K5Ac and H4K8 Ac,indicating that OGT1 mediated O-GlcNAcylation of NSL3 may regulate the holoenzyme activity of MOF/NSL complex.But the molecular mechanism still remains unclear.We discovered that OGT1 enhanced the ubiquitin-conjugating enzyme UBE2 S regulated stability of NSL3 through O-GlcNAc modifying its C-Terminus via in vivo/vitro O-GlcNAc assay using NSL3 truncated mutants.Furthermore,mass spectrometry analysis was performed and 6 potential O-GlcNAc modification sites on C-terminus of NSL3 were presumed.A point mutation plasmid for each site was constructed for O-GlcNAc assay and the result indicated that T755 was a major O-GlcNAcylation site.What counts was that the O-GlcNAc modification of this site remarkably weakened the interaction between NSL3 and UBE2 S thus inhibit the ubiquitin-proteasome degradation of NSL3 and enhanced its protein stability.Moreover,the O-GlcNAc modification of NSL3 Thr755 site by OGT1 not only stabilized NSL3,but more importantly,enhanced the structural stability and integrity and then increased the holoenzyme activity of NSL complex directly.As for the role of OGT1 mediated biological function in cells,colony formation of A549 cells as a preliminary study was performed,which clarified that the O-GlcNAc modification of NSL3 increased the proliferation and colony formation of A549 cells.Taken together,the integrity of MOF/NSL complex was required for cancer cell proliferation.