Regulation and readout of mammalian DNA methylation

Abstract

The mesmerizing phenotypical and functional diversity of mammalian cell types is to a large extent attributed to epigenetic signals that work together with the DNA sequence to determine gene expression programs. DNA methylation is one of the most important types of epigenetic signals and its paramount role was recognized in early genetic studies. Still, even after decades of active research, a comprehensive understanding of the mechanisms that regulate the chromatin targeting and activity of DNA methyltransferases has not been achieved. In this work, three main directions of research were undertaken, with the ultimate goal of shedding mechanistic and methodological insights into the generation and maintenance of DNA methylation patterns. In the first project of this thesis, a combination of biochemical and cellular experiments was used to assess the cellular role of the putative chromatin remodeler HELLS, an essential cofactor of DNA methyltransferases. By employing chromatin fractionation assays and microscopy-based techniques, I could show that the ATPase activity of HELLS is necessary for the high nuclear mobility of the protein and its ability to get released from compacted chromatin sites. In addition, the H3K9me3 pathway was also found to play an important role in the exchange of HELLS at heterochromatin. Taken together, this work provides the first evidence for a role of ATP hydrolysis in the association between HELLS and chromatin and hints at a model where the fast exchange of HELLS at repetitive DNA sequences might enhance the local recruitment of epigenetic enzymes, such as DNA methyltransferases (DNMTs). This could subsequently lead to the local stabilization of silencing complexes at heterochromatin. In the second project of this thesis, the putative interaction between the de novo DNA methyltransferase DNMT3A and the 5mC-reading protein MeCP2 was addressed. By building on previous data from our laboratory, which documented a direct interaction between the TRD domain of MeCP2 and the ADD domain of DNMT3A, causing an inhibition of DNMT3A activity in vitro, I could show that these proteins also interact in the mouse brain and the inhibitory effect of this interaction is also observed in stable cells lines overexpressing MeCP2. Furthermore, by using conformationally locked DNMT3A variants as novel tools to study the allosteric regulation of this enzyme, I could elucidate the mechanism of the inhibition of DNMT3A by MeCP2. Accordingly, I found that MeCP2 stabilizes an allosterically closed conformation of DNMT3A, an effect that could be successfully relieved by addition of unmodified histone H3. These results were supported by whole genome bisulfite brain methylome analysis of a Mecp2 knockout mouse model. Collectively, the findings derived from this project offer unprecedented insights into the regulation of DNMT3A activity and propose a model in which the enzyme is under the combined control of MeCP2 and H3 tail modifications. Accordingly, depending on the modification status of the H3 tail at target sites, MeCP2 can act as either a repressor or activator of DNA methylation. Finally, in the third project of this thesis, the focus was placed on the development and application of a novel method that would enable for the first time the locus-specific visualization of epigenetic modifications in living mammalian cells. This urgent and unmet technological need was solved by developing a set of modular fluorescence complementation-based epigenetic biosensors for live cell microscopy applications. In these tools, the high DNA sequence specificity of engineered anchor proteins such as ZFs, TALEs, and CRISPR/Cas9 proteins, was combined with the great versatility of chromatin reading domains as natural detector modules of DNA methylation and histone 3 lysine 9 trimethylation. With this approach, I could detect both of these marks for the first time, at defined, endogenous DNA sequences in different mouse and human cell lines. Furthermore, I could follow the changes in the levels of these epigenetic modifications with locus-specific resolution after treatment with epigenetic inhibitors or the induction of epigenetic enzymes. It is anticipated that either in their present form or in combination with the ongoing developments in genomic targeting and microscopy technologies, these tools will greatly improve our understanding of how specific epigenetic signals, like DNA methylation, are set, erased and maintained during embryonic development or onset of disease. Taken together, the results of this doctoral thesis demonstrate how a synergistic use of biochemical and cellular methods allows to derive deep insights into the epigenetic signaling network centered around the regulation of mammalian DNA methylation.