Role and regulation of TET-mediated DNA modifications in gene expression

In the mammalian genome, cytosine methylation (5mC) plays a central role in the epigenetic regulation of gene expression and has been implicated in a variety of biological processes, including genome stability, imprinting or differentiation. Compared to other epigenetic marks, DNA methylation has been thought to be relatively stable. However, genome-wide loss of 5mC, or DNA demethylation, has been observed in specific developmental stages and in various types of cancer. The discovery of the TET family of enzymes in 2009 was a watershed moment in comprehending the mechanisms of DNA demethylation. TET proteins oxidize 5mC to 5- hydroxymethylcytosine (5hmC), 5-formlycytosine (5fC) and 5-carboxylcytosine (5caC), which not only serve as key intermediates in active DNA demethylation pathways, but can also act as independent epigenetic marks. In this study, various aspects of TET-mediated DNA demethylation have been intensively investigated. Using quantitative mass-spectrometry-based proteomics readers for the different cytosine derivatives in mouse embryonic stem cells (ESCs), neuronal progenitor cells, and adult mouse brain tissue were identified. Readers for these modifications are only partially overlapping and are dynamic during differentiation. Moreover, the oxidized derivatives of 5mC recruit distinct transcription regulators as well as a large number of DNA repair proteins, implicating DNA damage response as the main pathway contributing to active DNA demethylation. To identify additional non-canonical DNA bases, highly sensitive quantitative mass-spectrometry led to the discovery of 5-hydroxymethyluracil (5hmU) in ESCs. Genomic 5hmU is not generated via deamination of 5hmC, as widely suggested, but through direct oxidation of thymine by TET proteins. In addition, screening for specific 5hmU readers identified different transcriptional and epigenetic factors, implicating that this mark has a specific function in ESCs. So far, only little is known how TET enzymes are regulated and how they are modified by posttranslational modifications (PTMs). Mapping TET phosphorylation and glycosylation sites at amino acid resolution revealed that these PTMs are interdependent and mostly occur at regulatory protein regions. Finally, a reporter gene based assay could demonstrate that in vitro methylation causes gene silencing while subsequent oxidation, resulting in DNA demethylation, leads to gene reactivation in vivo. Different knockout and rescue experiments clearly show that oxidation of methylcytosine by TET proteins and subsequent removal by TDG or NEIL glycosylases and the base excision repair pathway results in reactivation of epigenetically silenced genes. In conclusion, this work provides new insights how TET proteins can set DNA modifications, how these oxidized bases are read by various factors and how TET proteins can be posttranslationally modified. Furthermore, removal of 5mC is achieved through TET-mediated oxidation and depends on the activity of specific glycosylases, which leads to gene reactivation