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Abstract

O-GlcNAcylation is a reversible posttranslational modification (PTM) found on thousands of nuclear and cytoplasmic proteins catalyzed by OGT and OGA enzymes. Because of the embryonic and cellular lethality resulting from Ogt mutation, the mammalian function of OGlcNAc modification is still unknown. Despite the extensive studies since 1987, when OGlcNAcylation was identified for the first time, the role of O-GlcNAc and OGT in regulating gene expression remains still enigmatic. With my PhD project I aim at studying the role of O-GlcNAc modification in mouse chromatin by combining genomic and proteomic approaches. Pan-GlcNAc chromatin profiling revealed that O-GlcNAc proteins densely occupy promoter regions of pluripotent and neuronal differentiated cells. Unbiased bioinformatic screening for co-occupancy with public ChIP-seq data sets revealed that O-GlcNAc strongly co-localizes with RNA Polymerase II (RNA Pol II) at promoters. RNA Pol II was previously shown to be modified by O-GlcNAc modification; while it is well described how RNA Pol II catalyzes the DNA-directed mRNA synthesis of proteincoding genes, the mechanism by which O-GlcNAc regulates its activity is still unknown. OGlcNAc nuclear perturbation followed by super-resolution imaging revealed a novel role for OGlcNAc modification in regulating RNA Pol II localization at nuclear transcription factories. To gain deeper insight into O-GlcNAc mechanism in modulating RNA Pol II activity, I have established new tools to perturb and detect O-GlcNAc modification selectively on the Cterminal domain (CTD) of RNA Pol II. To investigate how O-GlcNAc regulates transcription at promoter regions, we have engineered and validated a novel inducible perturbation system to deplete O-GlcNAc at CpG dense promoters in pluripotent and differentiated cells. Our transcriptomics analysis revealed that targeted O-GlcNAc depletion at CpG-dense promoters causes the perturbation of several metabolic and mitochondrial genes, as well as a significant upregulation in ribosomal subunits expression. qPCR analysis of rRNA demonstrated that O-GlcNAc perturbation at CpG-rich sites leads to a strong deregulation of rRNA expression. Overall, I identified a novel function for O-GlcNAc in the regulation of ribosome biogenesis. This finding is consistent with my proteomic data obtained from the characterization of the OGT interactome, which revealed that nuclear OGT strongly interacts with a large number of nucleolar and ribosomal subunits. Preliminary mass spectrometry analysis of the nuclear O-GlcNAc proteome showed that the majority of nuclear O-GlcNAc proteins are factors involved in RNA Pol I and III transcriptional regulation, the two RNA Polymerases that control rRNA transcription. Taken together, my results demonstrate how O-GlcNAc chromatin proteins are not uniformly distributed all over the genome, but instead occupy predominantly gene promoters: part of these promoters are transcriptionally silenced, while the majority of them are active genes. Chromatin enrichment analysis revealed that O-GlcNAc highly correlates with RNA Pol II occupancy, suggesting that O-GlcNAc might regulate the expression of RNA Pol II-rich genes. Finally, by transcriptomics and proteomics approaches I have described a novel mechanism of nuclear O-GlcNAcylation: my analysis demonstrated how nuclear OGT regulates, via OGlcNAc modification, ribosomal biogenesis by controlling the expression of ribosomal proteins and of rRNA. These results bring new avenue into the possible mechanism of OGT in adapting the cellular metabolic state in response to the intracellular O-GlcNAc levels.