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.