Ten-Eleven-Translocation (TET) proteins form a family of enzymes responsible for active DNA demethylation by oxidation of 5-methylcytosine. TET proteins play a key role in genomic reprogramming in vitro and in vivo. Although TET proteins are expressed in embryonic stem cells (ESCs), their role in regulating pluripotency remains unclear. In addition, the mechanisms by which TET proteins are recruited to chromatin are largely unknown. To visualise TET protein dynamics during pluripotency and differentiation, the endogenous Tet1/2/3 alleles were fused to epitope tags in ESCs using CRISPR/Cas9. Characterisation of these cell lines showed that TET1 is the highest expressed TET protein in both naïve and primed pluripotent cells. In contrast, TET2 is expressed heterogeneously in ESCs and marks cells with a high self-renewal capacity. To assess the function of Tet genes in pluripotent stem cells, the endogenous Tet1/2/3 ORFs were removed using CRISPR/Cas9. Comparative analysis of single and combined Tet gene knockout ESC lines indicated that Tet1 and Tet2, but not Tet3, play redundant roles to promote loss of pluripotency. Furthermore, Tet-deficient cells retained a naïve morphology in differentiating conditions, suggestive of a LIF-independent self-renewal phenotype. To characterise physiological TET1 protein-protein interactions, TET1 protein partners were identified in ESCs by mass spectrometry and co-immuno-precipitations. This revealed that TET1 interacts with multiple epigenetic and pluripotency-related factors in ESCs. Moreover, detailed characterisation of the interaction between TET1 and NANOG identified three regions of TET1 involved in protein-protein interactions that are conserved in evolution. To investigate TET1 chromatin binding in ESCs, both at the molecular and cellular levels, TET1 was characterised by ChIP-seq analysis and live imaging experiments. Interestingly, TET1 is targeted to chromatin by two different mechanisms, involving distinct protein regions. The interaction with multiple protein partners, including NANOG, might enable TET1 to be targeted to specific chromosomal locations. Additionally, TET1 has the unusual ability to bind mitotic chromatin through its N-terminus, independently of its interaction with NANOG. Together these analyses provide a new understanding of the role of TET proteins in pluripotent cells, as well as a detailed map of TET1 residues involved in protein-protein interactions and mitotic chromatin binding.

It has been known for a long time that the cytosine base can be modified to produce a new nucleotide, identified as 5-methylcytosine (mC). In normal cells, mC is correctly distributed into the genome, but in many diseases including life-threatening cancers, its pattern is profoundly perturbed. In 2009, Anjana Rao, published that certain proteins, known as the TET enzymes, are capable of removing mC by further oxidizing it to 5-hydroxymethylcytosine (hmC). This original article, cited more than 1200 times, has led to a great expansion in our understanding of DNA methylation. Such recent publications expanded this knowledge by showing that the TETs successively oxidize hmC to 5-formylcytosine (fC) and 5-carboxylcytosine (caC). <p>These oxidized methylcytosines have been implicated in several mechanisms of DNA demethylation, including “active” demethylation through base excision repair, and “passive” demethylation via successive rounds of DNA replication. In addition, DNA hydroxymethylation is thought to be involved in a wide range of diseases, and a marked decrease of hmC seems to be a “hallmark” of many cancers. <p>However, little is known about the regulation of their modes of action. It is tempting to speculate that these proteins interact with a plethora of factors to elicit coordinated biological functions. Likewise, they might be regulated by environment, which in certain situations, could alter the hydroxymethylome landscape, and lead to cellular malfunction and diseases.<p>In the first study, we pursued a large, unbiased screen of the TET interactome, and discovered that TET2 and TET3 interact with the O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT). OGT is a glycosyltransferase that adds N-acetylglucose moieties on various proteins, including histone H2B, expanding therefore the “histone code”. We further discovered that the TET-OGT association seems to enhance OGT activity and to potentiate glycosylation and stabilization of SET1/COMPASS, a complex that is responsible for the global deposition of the H3K4me3 histone mark that “decorates” active promoters. Finally, we could confirm a decreased genome-wide H3K4me3 deposition in a model of acute myeloid leukemia mutated for TET2, suggesting that the TET-OGT link is implicated in Health and Disease.<p>In the second study, we looked at the impact of the environment on TET activity and on cellular hydroxymethylomes. We focused on oxidative stress assaults that are known to be involved in inflammation, a mediator of cancer and neurodegenerative diseases. We observed a significant decrease of hmC in cell lines treated with various oxidant stressors, likely due to a direct inactivation of the TETs catalytic domain. Moreover, gene ontology analysis of differentially hydroxymethylated regions (dhMRs), profiled by deep-sequencing on treated vs non-treated cells, highlighted pathways involved in oxidative stress response. The implication of TETs in oxidative stress response was further emphasized by a decreased proliferation of TET1-depleted cells when they are treated with oxidant stressors. Importantly, those results were confirmed in mice knockout for the major antioxidant enzymes GPx1 and GPx2. <p>In conclusion, the work of this thesis contributed to better understand the modes of action of the TET proteins, through (1) direct interaction with OGT, and (2) via direct regulation by oxidative-stress-associated molecules, and we hope that these results will bring new insights to better understand these fascinating enzymes. <p> / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

Antibiotic resistance is an ongoing global medical crisis and we are in great need of new antibacterial agents to combat rapidly emerging resistant pathogens. Tigecycline is one of few drugs that have been introduced into medicine during the last two decades. It is a broad-spectrum third generation tetracycline that is active against multidrug-resistant bacteria that cause complicated infections. In this thesis I examined the development of tigecycline resistance in Escherichia coli and associated in vitro and in vivo fitness effects. Selections of spontaneous E. coli mutants revealed relatively high accumulation rates of changes in the multidrug efflux system AcrAB-TolC regulation network and in heptose biosynthesis and transport pathways important for lipopolysaccharide (LPS) synthesis. Both groups of mutations led to reduced susceptibility to tigecycline and slower growth compared to the wild-type bacteria. Additional in vitro fitness assays and in vivo competitions showed that LPS mutants were less fit than efflux mutants, providing a possible explanation for why up-regulation of multidrug efflux pumps is the main tigecycline resistance mechanism reported in clinical isolates. Tigecycline was designed to evade the two most common tetracycline resistance mechanisms conferred by Tet proteins, efflux and ribosomal protection. However, tigecycline is a substrate for the tetracycline modifying enzyme Tet(X). Screening of Tet protein mutant libraries showed that it is possible to select Tet mutants with minimal inhibitory concentrations of tigecycline that reach clinically relevant levels. Mutations in Tet proteins that permitted a better protection from tigecycline frequently exhibited reduced activity against earlier generations of tetracyclines, except for the Tet(X) enzyme mutants, which were better at inactivating all tested tetracyclines. This is particularly worrisome because different variants of Tet(X) have recently spread to multidrug-resistant pathogens through horizontal gene transfer. Therefore, Tet(X) mutants with improved activity threaten the medical future of tetracyclines. Multidrug resistance is easily disseminated through horizontally spreading conjugative plasmids. pUUH239.2 is an example of a successful conjugative plasmid that caused the first clonal outbreak of extended spectrum β-lactamase-producing Klebsiella pneumoniae in Scandinavia. This plasmid was formed after rearrangements between two different plasmid backbones and it carries resistance genes to multiple antibiotic classes, heavy metals, and detergents.