Enrichment Analysis Of O-GlcNac Modified Chromatin Binding Sites In Breast Cancer Cells Based On DNase-seq

O-linked N-acetylglucosamine modification(O-GlcNAc)is an important posttranslational modification that occurs widely on nuclear and cytoplasmic proteins.O-GlcNAc has numerous glycosylation substrates and is involved in the regulation of almost all physiological and pathological processes.Studies have shown that there are a large number of O-GlcNAc modified proteins on chromatin,which affect the biological function of chromatin.The previous research results of this subject suggest that the O-GlcNAc modification of transcription factor regulatory proteins bound to chromatin plays an important role in the regulation of gene transcription.O-GlcNAc glycosylation can be enriched in open regions of chromatin,affecting transcriptional activity of transcription factors,DNA binding,nuclear localization,stability,and interactions with other cofactors.Therefore,it is of great significance to study the binding law of glycosylation to transcription factors in the open region of chromatin for in-depth analysis of the regulation of transcription.In this paper,we combined the DNase-seq technology to study chromatin open regions with the O-GlcNAc metabolic labeling strategy,and developed a genome-wide method to enrich and analyze O-GlcNAc glycosylation binding of chromatin open regions(OGTFDNase-seq).In this method,after O-GlcNAc glycometabolism labeling,the labeled chromatinbinding protein was first fixed with DNA using formaldehyde cross-linking,and then the nuclei were isolated and the open chromatin region was subjected to hypersensitive DNase I digestion,thereby obtaining small fragments of the open region of chromatin.Then,the biotin was linked to the azide-labeled chromatin fragment through the Click reaction,and the O-GlcNAc modified open chromatin complex was affinity precipitated by streptavidin magnetic beads and biotin.Finally,the DNA fragments were reverse-crosslinked and released,and after extraction and purification,the enriched DNA fragments were used for high-throughput sequencing analysis,so as to achieve accurate identification of the chromatin open regions bound by OGlcNAc glycosylation.Further,we performed a bioinformatic analysis of the OGTF-DNase-seq sequencing data.After sequence alignment and peak calling analysis of enriched sites,the results showed that in breast cancer cell MCF-7 and the corresponding multidrug-resistant cell MCF-7/ADR samples,the distribution of O-GlcNAc chromatin-bound open regions in the human genome was significantly different.Although both MCF-7 and MCF-7/ADR signals were mainly distributed at the gene transcription start site(TSS),the signal was more concentrated at the TSS in drugresistant cells,suggesting that O-GlcNAc glycosylation was in MCF-7/ ADR cells were more involved in genome transcriptional regulation.The results of association analysis with transcriptomics(RNA-seq)showed that most of the 4252 O-GlcNAc glycosylation-bound downstream genes in the chromatin open region were transcriptionally activated rather than repressed,suggesting that O-GlcNAc modified chromatin regulators had transcriptional activation effect.The functional enrichment analysis of these genes suggested that signaling pathways such as cell adhesion and migration,cellular stress response,and regulation of DNAbinding transcription factor activity might be involved in the O-GlcNAc-regulated drug resistance process in breast cancer cells.In conclusion,here we developed a method to enrich the open regions of chromatin bound by O-GlcNAc glycosylation.O-GlcNAc glycosylation in breast cancer cells participated in gene transcription activation by regulating the open region of chromatin,affected the expression of various downstream malignant transformation signaling genes,and thus participated in the regulation of tumor drug resistance.