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Schmidt, Christine Silvia (2012): The role of DNA modifications in pluripotency and differentiation. Dissertation, LMU München: Fakultät für Biologie



DNA methylation plays a crucial role in the epigenetic control of gene expression during mammalian development and differentiation. Whereas the de novo DNA methyltransferases (Dnmts), Dnmt3a and Dnmt3b, establish DNA methylation patterns during development; Dnmt1 stably maintains DNA methylation patterns during replication. DNA methylation patterns change dynamically during development and lineage specification, yet very little is known about how DNA methylation affects gene expression profiles upon differentiation. Therefore, we determined genome-wide expression profiles during differentiation of severely hypomethylated embryonic stem cells (ESCs) lacking either the maintenance enzyme Dnmt1 (dnmt1-/- ESCs) or all three major Dnmts (dnmt1-/-; dnmt3a-/-, dnmt3b-/- or TKO ESCs), resulting in complete loss of DNA methylation, and assayed their potential to transit in and out of the ESC state. Our results clearly demonstrate that upon initial differentiation to embryoid bodies (EBs), wild type, dnmt1-/- and TKO cells are able to activate differentiation processes. However, transcription profiles of dnmt1-/- and TKO EBs progressively diverge with prolonged EB culture, with dnmt1-/- EBs being more similar to wild type EBs, indicating a higher differentiation potential of dnmt1-/- EBs compared to TKO EBs. Remarkably though, after dissociation of late EBs and further cultivation under pluripotency promoting conditions, both dnmt1-/- and TKO but not wild type cells rapidly revert to expression profiles typical of undifferentiated ESCs. Thus, while DNA methylation is dispensable for the initial activation of differentiation programs, it seems to be crucial for permanently restricting the developmental fate during differentiation. Based on the essential role of Uhrf1 in maintenance DNA methylation, we investigated the structurally highly similar second member of the Uhrf protein family, Uhrf2, whose function in maintenance methylation or other biological processes is completely unknown. Expression analysis of uhrf1 and uhrf2 in various cell lines and tissues revealed a time- and developmental switch in transcript levels of both genes with uhrf1 being highly expressed in undifferentiated, proliferating cells and uhrf2 being predominately expressed in differentiated, non-dividing cells. These opposite expression patterns together with no detectable effect on DNA methylation levels upon knock down of uhrf2 suggests that Uhrf2 is rather involved in maintaining DNA methylation patterns in differentiated cells and points to non-redundant functions of both proteins. The discovery of the “6th base” of the genome, 5-hydroxymethylcytosine (5hmC), resulting from the oxidation of 5mC by the family of Tet dioxygenases (Tet1-3), once again ignited the debate about how DNA methylation marks can be modified and removed. To gain insights into the biological function of this newly identified modification, we developed a sensitive enzymatic assay for quantification of global 5hmC levels in genomic DNA. Similar to 5mC levels, we found that also 5hmC levels dynamically change during differentiation of ESCs to EBs, which correlates with the differential expression of tet1-3. Furthermore, we characterized a novel endonuclease, PvuRts1I that selectively cleaves 5hmC containing DNA and show first data on its application as a tool to map and analyze 5hmC patterns in mammalian genomes. Finally, we investigated designer transcription activator-like effector (dTALEs) proteins targeting the oct4 locus. Our results show that the epigenetic state of the target locus interferes with the ability of dTALEs to activate transcriptionally silent genes, which however can be overcome using dTALEs in combination with low doses of epigenetic inhibitors. In conclusion, this work gives further insights into the biological roles of methylation mark writers (Dnmts), readers (Uhrfs) and modifiers (Tets) and advances our understanding on the function of DNA methylation in the epigenetic control of gene expression during development and cellular differentiation.