The role of DNA modifications in pluripotency and differentiation

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.