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Abstract

O-GlcNAcylation (O-GlcNAc) is a post-translational modification found on serine and threonine residues of a variety of nuclear and cytosolic mammalian proteins. In spite of the diversity of its targets, this modification is catalyzed by one single enzyme known as O-GlcNAc transferase (OGT), which is highly conserved in animals. In mammals, a maternal functional copy of the Ogt gene is essential for embryonic development to postimplantation stages, indicating an important function of OGT in mammalian early development. An ever-increasing number of in vitro studies report functions of OGT and O-GlcNAc on many cellular pathways, spanning from the cell cycle to the regulation of transcription. Nuclear factors reported to bear O-GlcNAc moieties include RNA Polymerase II and the transcription factors OCT4 and SOX2, which are master regulators of pluripotency. These findings, together with the early lethality caused by the lack of maternal OGT, raise the intriguing possibility that OGT and O-GlcNAc might be involved in transcriptional regulation during early mammalian development. Furthermore, the donor substrate for O-GlcNAcylation is UDP-GlcNAc, the end product of one of the metabolic routes of intracellular glucose. This could in principle make O-GlcNAc levels responsive to changes in the environmental conditions or intracellular metabolic demands, hence a potential mediator of cellular adaptation to such changes. Due to the difficulty of disrupting mammalian Ogt using classical genetic approaches, these intriguing hypotheses have never been tested in vivo. With my study, I probed the effect of O- GlcNAc perturbation on the developing mammalian early embryo for the first time. To this end, I developed three different functional strategies using the mouse as a mammalian model organism. The first method is a degron system for a fast and inducible degradation of the endogenous OGT protein. This approach proved to be very efficacious in primary mouse embryonic fibroblasts (MEFs) in vitro, thus I used it to get a first insight into OGT-mediated transcriptional regulation. The transcriptome of OGT-depleted MEFs showed a low-magnitude but widespread change in gene expression, including a significant downregulation of the mesenchymal differentiation pathway and genes associated with translation. Nonetheless, the Ogt-degron system was very poorly effective when applied to the preimplantation embryo grown ex vivo, based on both O-GlcNAc staining and transcriptomics analyses. 5The second approach was the creation of a collection of Ogt hypomorphic mouse alleles, predicted to have a range of severity of phenotypes based on the catalytic activity of the corresponding OGT mutant proteins previously measured in vitro. I found that the severity of phenotypes of the different Ogt mutant mouse lines mirrored the level of in vitro activity reduction. In addition, unexpectedly, one of these mouse alleles showed a maternal effect phenotype: the genotype of the mother impacted the early embryo independently of the embryo’s genotype. Finally, I employed the overexpression of a recombinant OGA (the enzyme which removes O- GlcNAc) to remove the O-GlcNAc modification itself from the nucleus of the mouse zygote. By injecting the mRNA of OGA in the zygote, O-GlcNAc was reduced to undetectable levels from the early 2-cell stage, before the activation of the embryonic genome. This allowed me to specifically test the role of O-GlcNAc in genome regulation during preimplantation. I discovered that nuclear O-GlcNAc is largely indispensable for embryonic genome activation, but that O-GlcNAc depletion leads to a subtle preimplantation developmental delay visible in the morula only at the transcriptome level. When embryos developed from O-GlcNAc-depleted zygotes were re-implanted into a foster mother and dissected postimplantation, they were developmentally delayed. Based on my transcriptome data, I propose possible molecular mechanisms that could explain this phenotype. One is the effect of O-GlcNAc perturbation on the cell cycle, another is the modulation of the rate of protein synthesis by O-GlcNAc. In sum, my PhD work brings novel insights into the biological function of the still enigmatic O-GlcNAc modification in the early mammalian embryo.