<> "The repository administrator has not yet configured an RDF license."^^ . <> . . "The role of a nuclear glycosylation in the early mammalian embryo."^^ . "O-GlcNAcylation (O-GlcNAc) is a post-translational modification found on serine and\r\nthreonine residues of a variety of nuclear and cytosolic mammalian proteins. In spite of the\r\ndiversity of its targets, this modification is catalyzed by one single enzyme known as O-GlcNAc\r\ntransferase (OGT), which is highly conserved in animals. In mammals, a maternal functional\r\ncopy of the Ogt gene is essential for embryonic development to postimplantation stages,\r\nindicating an important function of OGT in mammalian early development. An ever-increasing\r\nnumber of in vitro studies report functions of OGT and O-GlcNAc on many cellular pathways,\r\nspanning from the cell cycle to the regulation of transcription. Nuclear factors reported to bear\r\nO-GlcNAc moieties include RNA Polymerase II and the transcription factors OCT4 and SOX2,\r\nwhich are master regulators of pluripotency. These findings, together with the early lethality\r\ncaused by the lack of maternal OGT, raise the intriguing possibility that OGT and O-GlcNAc\r\nmight be involved in transcriptional regulation during early mammalian development.\r\nFurthermore, the donor substrate for O-GlcNAcylation is UDP-GlcNAc, the end product of one\r\nof the metabolic routes of intracellular glucose. This could in principle make O-GlcNAc levels\r\nresponsive to changes in the environmental conditions or intracellular metabolic demands,\r\nhence a potential mediator of cellular adaptation to such changes.\r\nDue to the difficulty of disrupting mammalian Ogt using classical genetic approaches, these\r\nintriguing hypotheses have never been tested in vivo. With my study, I probed the effect of O-\r\nGlcNAc perturbation on the developing mammalian early embryo for the first time. To this end,\r\nI developed three different functional strategies using the mouse as a mammalian model\r\norganism.\r\nThe first method is a degron system for a fast and inducible degradation of the endogenous\r\nOGT protein. This approach proved to be very efficacious in primary mouse embryonic\r\nfibroblasts (MEFs) in vitro, thus I used it to get a first insight into OGT-mediated transcriptional\r\nregulation. The transcriptome of OGT-depleted MEFs showed a low-magnitude but widespread\r\nchange in gene expression, including a significant downregulation of the mesenchymal\r\ndifferentiation pathway and genes associated with translation. Nonetheless, the Ogt-degron\r\nsystem was very poorly effective when applied to the preimplantation embryo grown ex vivo,\r\nbased on both O-GlcNAc staining and transcriptomics analyses.\r\n5The second approach was the creation of a collection of Ogt hypomorphic mouse alleles,\r\npredicted to have a range of severity of phenotypes based on the catalytic activity of the\r\ncorresponding OGT mutant proteins previously measured in vitro. I found that the severity of\r\nphenotypes of the different Ogt mutant mouse lines mirrored the level of in vitro activity\r\nreduction. In addition, unexpectedly, one of these mouse alleles showed a maternal effect\r\nphenotype: the genotype of the mother impacted the early embryo independently of the\r\nembryo’s genotype.\r\nFinally, I employed the overexpression of a recombinant OGA (the enzyme which removes O-\r\nGlcNAc) to remove the O-GlcNAc modification itself from the nucleus of the mouse zygote.\r\nBy injecting the mRNA of OGA in the zygote, O-GlcNAc was reduced to undetectable levels\r\nfrom the early 2-cell stage, before the activation of the embryonic genome. This allowed me to\r\nspecifically test the role of O-GlcNAc in genome regulation during preimplantation. I\r\ndiscovered that nuclear O-GlcNAc is largely indispensable for embryonic genome activation,\r\nbut that O-GlcNAc depletion leads to a subtle preimplantation developmental delay visible in\r\nthe morula only at the transcriptome level. When embryos developed from O-GlcNAc-depleted\r\nzygotes were re-implanted into a foster mother and dissected postimplantation, they were\r\ndevelopmentally delayed. Based on my transcriptome data, I propose possible molecular\r\nmechanisms that could explain this phenotype. One is the effect of O-GlcNAc perturbation on\r\nthe cell cycle, another is the modulation of the rate of protein synthesis by O-GlcNAc.\r\nIn sum, my PhD work brings novel insights into the biological function of the still enigmatic\r\nO-GlcNAc modification in the early mammalian embryo."^^ . "2024" . . . . . . . "Sara"^^ . "Formichetti"^^ . "Sara Formichetti"^^ . . . . . . "The role of a nuclear glycosylation in the early mammalian embryo. (Other)"^^ . . . . . . "indexcodes.txt"^^ . . . "The role of a nuclear glycosylation in the early mammalian embryo. (PDF)"^^ . . . "The role of a nuclear glycosylation in the early mammalian embryo. (Other)"^^ . . . . . . "lightbox.jpg"^^ . . . "The role of a nuclear glycosylation in the early mammalian embryo. (Other)"^^ . . . . . . "preview.jpg"^^ . . . "The role of a nuclear glycosylation in the early mammalian embryo. (Other)"^^ . . . . . . "medium.jpg"^^ . . . "The role of a nuclear glycosylation in the early mammalian embryo. (Other)"^^ . . . . . . "small.jpg"^^ . . "HTML Summary of #33813 \n\nThe role of a nuclear glycosylation in the early mammalian embryo.\n\n" . "text/html" . . . "570 Biowissenschaften, Biologie"@de . "570 Life sciences"@en . .