Application and engineering of targeted DNA methylation editing tools for the modulation and characterization of the epigenome network

Abstract

Despite of the large variety of different cell types in humans, all cells carry the same genetic information. In order to enable a cell type-specific expression profile, a multitude of epigenetic signals has to be established and maintained by various epigenetic mechanisms. Histone post-translational modifications and DNA methylation are two major epigenetic signals and parts of the epigenome network, which regulate the activation or silencing of gene expression by modulating the chromatin accessibility. In mammals, DNA methylation patterns are established by DNA methyltransferases, and aberrant methylation patterns have been connected to various disorders including cancer. Thus, it is of particular interest for basic research and medical applications to gain a better understanding of this mark, and to artificially manipulate the DNA methylation state at a certain locus. This can be achieved by the employment of diverse epigenome editing tools, termed ‘EpiEditors’, allowing to install or remove DNA methylation with variable specificity. Aim of the main part of this PhD work was to characterize the stability of DNA methylation at CpG islands (CGIs) after artificial introduction. For installation of DNA methylation, a zinc finger (ZnF) was used as targeting device, which has a short DNA binding motif allowing to target numerous genomic loci simultaneously. By conducting ChIP experiments, more than 15,000 ZnF peaks were identified, demonstrating its promiscuous binding in the genome. The fusion of the DNMT3A catalytic domain (3AC) to the ZnF enabled to deposit DNA methylation at thousands of CGIs. Afterwards, the dynamics of this mark were tracked for up to 11 days by MBD2-seq. Strikingly, the installed DNA methylation was only transient at around 90 % of all previously unmethylated CGIs. However, high levels of residual DNA methylation were observed at 10 % of the CGIs. Intriguingly, these stably methylated CGIs were strongly enriched in H3K27me3, a mark associated with Polycomb group chromatin. The deposition of DNA methylation resulted in a marked reduction of the activating marks H3K4me3 and H3K27ac, which were partially recovered upon DNA methylation loss. Surprisingly, no direct effects of DNA methylation were observed on H3K9me3 and H3K36me3 levels. However, the expression of more than 900 genes was downregulated at least two-fold after DNA methylation editing of the corresponding promoter, demonstrating the high silencing capacity of the mark. Thereby, the expression changes were tightly correlated to the temporal changes promoter methylation levels. In the second project of this thesis, the goal was to develop an improved EpiEditor, capable to introduce DNA methylation with high efficiency at the target locus but with low off-target activity. For this, two dCas9-based targeting strategies in combination with two different effector domains, 3AC and a chimeric construct of 3AC fused to the C-terminal domain of DNMT3L (3AC-3L), were compared. The direct fusion of the effector domains to dCas9 resulted in a lower specificity than a connection of dCas9 and effector domain with a dCas9-SunTag system, which is based on the recruitment of multiple antibody-fused effector domains to an array of GCN4 peptides fused to dCas9. Although a high specificity of the dCas9-SunTag system in combination with 3AC was observed, the DNA methylation efficiency was rather low. In contrast, dCas9-SunTag together with 3AC-3L was more efficient, but on genome-wide scale, the off-target activity was very high. By introducing charge-reversal mutations into basic residues required for the interaction of 3AC-3L with DNA, most of the off-target activity of the EpiEditor could be removed without compromising its on-target activity strongly. In the end, the novel EpiEditor outperformed the previously available dCas9-SunTag construct in combination with 3AC. The third project of this work aimed to reprogram the H19/IGF2 imprinting control region (ICR) by targeted DNA demethylation. To this end, the dCas9-SunTag system in combination with the catalytic domain of TET1 was employed. Multiple CpG-rich motifs within the ICR were targeted by using a multi-sgRNA plasmid. Strikingly, a strong reduction in DNA methylation was obtained. By conducting CTCF-ChIP, it could be demonstrated that the DNA demethylation was accompanied by the recruitment of the methylation-sensitive insulator protein CTCF. The reduced DNA methylation and the increased CTCF occupancy were maintained for almost four weeks, indicating the robustness of the reprogrammed state. All in all, the obtained data provide valuable information on the dynamics of targeted DNA methylation and its regulatory effects. In combination with the development of better EpiEditors, this PhD work may pave the way towards safer and more efficient clinical applications of EpiEditing.