Regulation of the TET1 protein in active DNA demethylation

Active DNA demethylation plays an important role in various biological contexts, such as embryonic development and embryonic stem cell (ESC) differentiation. The Ten-Eleven Translocation (TET) family enzymes play a key role in this process, as they iteratively oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). 5fC and 5caC can subsequently be removed by Thymine DNA glycosylase (TDG) and unmethylated cytosine is restored by base excision repair (BER), completing the process of active DNA demethylation. Before the discovery of the role of TET enzymes in active DNA demethylation, the Growth arrest and DNA-damage-inducible protein 45 alpha (GADD45A) had already been implicated in active DNA demethylation, but its exact role remained unknown. Recent findings highlighted that GADD45A physically and functionally cooperates with the TET family member TET1 to mediate active DNA demethylation. However, the mechanism by which GADD45A enhances TET1 activity is yet poorly understood. In addition, how TET enzymes are targeted to genomic loci, especially to enhancers, remains largely unresolved. In fact, whereas TET binding sites are mostly found at promoters, the oxidation products of TET – 5hmC, 5fC and 5caC – are mostly enriched at enhancers. In this thesis, I explored two TET-related topics 1) TET1 regulation by GADD45A and 2) TET regulation by RAD21-dependent chromatin looping. First, I found that the GADD45A interactor Mitogen-activated protein kinase kinase kinase 4 (MAP3K4) is most likely not involved in enhancing TET1 activity in a GADD45A-dependent manner. Then, I demonstrated that GADD45A can affect TET1 post-translational modifications: GADD45A decreases the ubiquitination and SUMOylation of full-length TET1, while it enhances the SUMOylation of the catalytic domain of TET1 (TET1CD). Finally, I established that GADD45A stabilizes TET1 protein levels, putatively by preventing Calpain-mediated decay. Nevertheless, the detailed mechanism behind this ...