Inhaltszusammenfassung

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 b...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 stabilization remains to be resolved in the future. A proposed model by which TET enzymes are targeted to enhancers to oxidize 5mC is promoter-enhancer looping. Promoter-enhancer loops are established by the Cohesin complex and can be abolished by depleting one of its subunits, such as RAD21. To test this model, I investigated the distribution of 5fC in RAD21-depleted – and hence promoter-enhancer loop depleted – mouse ESCs (mESCs) and observed that the deposition of 5fC at enhancers is RAD21-loop independent. Although these results do not provide an ultimate explanation for the discrepancy between TET binding sites at promoters and the enrichment of 5hmC, 5fC and 5caC at enhancers, this analysis supports the conclusion that targeting of enhancer demethylation does not rely on RAD21-dependent chromatin loops.» weiterlesen» einklappen

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DDC Sachgruppe:
Biowissenschaften, Biologie